Eukaryotic mechanosensory transduction channel

ABSTRACT

The present invention provides, for the first time, nucleic acids encoding a eukaryotic mechanosensory transduction channel (MSC) protein. The proteins encoded by these nucleic acids form channels that can directly detect mechanical stimuli and convert them into electrical signals. These nucleic acids and the proteins they encode can be used as probes for sensory cells in animals, and can be used to diagnose and treat any of a number of human conditions involving inherited, casual, or environmentally-induced loss of mechanosensory transduction activity.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/392,812, filed Sep. 9, 1999, which is incorporated by referenceherein.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No.DC03160, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention provides isolated nucleic acid and amino acidsequences of a novel family of eukaryotic mechanosensory ion channelsthat are designated mechanosensory transduction channels (MSC).

BACKGROUND OF THE INVENTION

[0004] The ability to detect mechanical stimuli is an essential andprevalent characteristic of living organisms, and is found from bacteriato simple metazoans to the most complex of mammals. Indeed, the abilityto detect mechanical stimuli and convert them into electrical signalsforms the basis of many central aspects of animal life, such as lighttouch, heavy touch, proprioception, baroreception, balance, and thecrown jewel, hearing. Even the ability of cells to stop growing when incontact with neighboring cells is likely dependent on mechanicalstimuli. Not surprisingly, therefore, numerous human conditions resultat least in part from an inability to detect mechanical stimuli, such asMeniere's Disease, sensorineural deafness, blood pressure disorders, andvarious types of cancers.

[0005] In general, the variety of known mechanosensory modalities arethought to be mediated by mechanically-gated cation channels presentwithin the membrane of receptor cells. This view has come in large partfrom detailed studies into the physiology of mechanosensation usingvarious cell types involved in mechanosensory detection, such as thehair cells of the vertebrate inner ear, single-celled ciliates such asParamecium, or the sensory neurons of Drosophila (see, e.g., Kernan etal., Neuron 12:1195-1206 (1994)). In Drosophila, the dendrite of thesensory neuron is enclosed in a cavity filled with a specializedreceptor lymph, which is unusually rich in potassium ions, and isfunctionally equivalent to the potassium-rich endolymph of thevertebrate cochlea. These potassium ions produce a transepithelialpotential difference, with the apical side of the epithelium beingpositively charged. Mechanical stimulation of the bristle, which isadjacent to the sensory neuron, generates a mechanoreceptor potentialwithin the neuron, detectable as a negative deflection of thetransepithelial potential, which reflects the flow of cations from thereceptor lymph into the sensory neuron.

[0006] Activation of the hair cells of vertebrates also result in theinflux of cations into cells (see, e.g., Hudspeth, Nature, 341:397-404(1989)). Each hair cell has a number of specialized microvillarstructures, called stereocilia, whose deflection results in theactivation of a putative channel present on the surface of the cell.Interestingly, electrophysiological studies have suggested that thesecells contain a similar number of receptor channels as they dostereocilia, suggesting that perhaps each receptor channel is coupled toa single stereocilium. In addition, studies of the kinetics of hair-cellactivation have suggested that the putative mechanosensory receptors aredirectly stimulated by mechanical force, resulting in the direct openingof the channel without the involvement of second messengers.

[0007] Despite the great importance of mechanosensation for animalbehavior and health, and the detailed electrophysiological understandingthat has been gained from the above-described studies, almost nothing isknown about the molecular basis of mechanosensory detection ineukaryotes. Several mutations and distantly related molecules involvedin this process have, however, been found. In Drosophila, for example, anumber of mutations have been isolated that disrupt mechanoreception,resulting in a variety of phenotypes such as reduced locomotor activity,total uncoordination, and even death (Kernan et al., Neuron 12:1195-1206(1994)). Also, mutations have been identified in the nematode C. elegansthat result in a loss of sensitivity to gentle touch (reviewed inGarcia-Aanoveros & Corey, Ann. Rev. Neurosci. 20:567-594 (1997)). Inaddition, a prokaryotic mechanosensory channel has been identified(Sukarev et al., Nature 368:265-268 (1994)). Still, despite theseadvances, the principle molecule of the mechanosensory transductionprocess in eukaryotes, the mechanically gated channel, has yet to beisolated or identified.

[0008] The identification and isolation of eukaryotic mechanosensorytransduction channels would allow for the development of new methods ofpharmacological and genetic modulation of mechanosensory transductionpathways. For example, availability of mechanosensory transductionchannel proteins would permit screening for high-affinity agonists,antagonists, and modulators of mechanosensation in animals. Suchmolecules could then be used, e.g., in the pharmaceutical industry, totreat one or more of the many human conditions involving loss orhyperactivation of mechanosensation. In addition, the determination ofnucleotide and amino acid sequences of mechanosensory transductionchannels associated with a human condition would provide new tools forthe diagnosis and/or treatment, e.g., gene-based treatment, of thecondition.

SUMMARY OF THE INVENTION

[0009] The present invention provides for the first time nucleic acidsencoding a eukaryotic mechanosensory transduction protein. The nucleicacids and the polypeptides they encode are referred herein asmechanosensory channel (MSC) nucleic acids and proteins. In vivo, MSCproteins form mechanosensory transduction channels that play a centralrole in many critical processes such as hearing, proprioception, andtactile sensation.

[0010] In one aspect, the present invention provides an isolated nucleicacid encoding a mechanosensory transduction protein, the protein havingat least one of the following characteristics: (i) comprising greaterthan about 70% amino acid sequence identity to SEQ ID NO:2 or SEQ IDNO:4; (ii) comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii)specifically binding to polyclonal antibodies generated against apolypeptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4; wherein the protein does not comprise the polypeptide sequence ofSEQ ID NO:6.

[0011] In one embodiment, the nucleic acid encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. Inanother embodiment, the nucleic acid comprises a nucleotide sequence ofSEQ ID NO:1 or SEQ ID NO:3, but not SEQ ID NO:5.

[0012] In another embodiment, the nucleic acid selectively hybridizesunder moderately stringent wash conditions to a nucleic acid comprisinga nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. In anotherembodiment, the nucleic acid selectively hybridizes under stringent washconditions to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3, but not SEQ ID NO:5.

[0013] In another embodiment, the nucleic acid is amplified by primersthat selectively hybridize under stringent hybridization conditions tothe same sequence as degenerate primer sets encoding an amino acidsequence selected from the group consisting of: LDVLIENEQKEV (SEQ IDNO:7), HHLFGPWAIII (SEQ ID NO:8), and VLINLLIAMMSDTYQRIQ (SEQ ID NO:9).

[0014] In another embodiment, the nucleic acid is less than 120 kb. Inanother embodiment, the nucleic acid is less than 90 kb. In anotherembodiment, the nucleic acid is less than 60 kb. In another embodiment,the nucleic acid is less than 30 kb. In another embodiment, the nucleicacid is less than 10 kb. In another embodiment, the nucleic acidsequence encoding the MSC protein is isolated away from its genomicneighbors.

[0015] In another aspect, the present invention provides an expressioncassette comprising a nucleic acid encoding a mechanosensorytransduction protein, the protein having at least one of the followingcharacteristics: (i) comprising greater than about 70% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii) comprising anamino acid sequence selected from the group consisting of SEQ ID NO:7,SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically binding topolyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; wherein the proteindoes not comprise the polypeptide sequence of SEQ ID NO:6.

[0016] In another aspect, the present invention provides an isolatedeukaryotic cell comprising the expression cassette.

[0017] In one aspect, the present invention provides an isolated nucleicacid encoding an extracellular domain of a mechanosensory transductionprotein, the extracellular domain comprising greater than about 70%amino acid sequence identity to an extracellular domain of SEQ ID NO:2or SEQ ID NO:4, wherein the extracellular domain does not comprise anextracellular domain of SEQ ID NO:6.

[0018] In one embodiment, the extracellular domain is fused to aheterologous polypeptide, thereby forming a chimeric polypeptide. Inanother embodiment, the extracellular domain comprises an amino acidsequence of an extracellular domain of SEQ ID NO:2 or SEQ ID NO:4.

[0019] In another aspect, the present invention provides an isolatedechanosensory transduction protein, the protein having at least one ofthe following haracteristics: (i) comprising greater than about 70%amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically bindingto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:2, or SEQ ID NO:4; wherein the proteindoes not comprise the amino acid sequence of SEQ ID NO:6.

[0020] In one embodiment, the protein comprises the amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:4.

[0021] In another aspect, the present invention provides an isolatedpolypeptide comprising an extracellular domain of a mechanosensorytransduction protein, the extracellular domain comprising greater thanabout 70% amino acid sequence identity to an extracellular domain of SEQID NO:2 or SEQ ID NO:4, wherein the extracellular domain does notcomprise the amino acid sequence of an extracellular domain of SEQ IDNO:6.

[0022] In one embodiment, the extracellular domain is fused to aheterologous polypeptide, forming a chimeric polypeptide. In anotherembodiment, the extracellular domain comprises the amino acid sequenceof an extracellular domain of SEQ ID NO:2 or SEQ ID NO:4.

[0023] In another aspect, the present invention provides an antibodythat selectively binds to a mechanosensory transduction protein, theprotein having at least one of the following characteristics: (i)comprising greater than about 70% amino acid sequence identity to SEQ IDNO:2 or SEQ ID NO:4; (ii) comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9;or (iii) specifically binding to polyclonal antibodies generated againsta polypeptide comprising an amino acid sequence of SEQ ID NO:2, or SEQID NO:4; wherein the protein does not comprise the amino acid sequenceof SEQ ID NO:6.

[0024] In another aspect, the present invention provides a method foridentifying a compound that modulates mechanosensory receptor activityin eukaryotic cells, the method comprising the steps of: (i) contactingthe compound with a mechanosensory receptor protein, the protein havingat least one of the following characteristics: (a) comprising greaterthan about 70% amino acid sequence identity to a sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; (b)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (c) specifically bindingto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, and SEQ ID NO:6; and (ii) determining the functional effectof the compound on the mechanosensory receptor protein.

[0025] In one embodiment, the mechanosensory receptor protein isexpressed in a eukaryotic cell or cell membrane. In another embodiment,the functional effect is determined by detecting a change in themechanoreceptor potential of the cell or cell membrane. In anotherembodiment, the functional effect is determined by detecting a change inan intracellular ion concentration. In another embodiment, the ion isselected from the group consisting of K⁺ and Ca²⁺. In anotherembodiment, the protein comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. Inanother embodiment, the protein is recombinant. In another embodiment,the functional effect is a physical interaction with the receptorprotein.

[0026] In another aspect, the present invention provides a method ofgenotyping a human for a mechanosensory transduction channel locus, themethod comprising detecting a mutation in a nucleic acid encoding amechanosensory transduction channel in the human, the protein having atleast one of the following characteristics: (a) comprising greater thanabout 70% amino acid sequence identity to a polypeptide having asequence of SEQ ID NO:2; (b) having greater than about 90% amino acidsequence identity to a polypeptide having a sequence of SEQ ID NO:5; (c)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; or (d) specifically bindingto polyclonal antibodies generated against a polypeptide selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:8; wherein the mutation introduces a premature stopcodon into the nucleic acid 5′ to the transmembrane domain region of theprotein, or is a missense mutation removing a cysteine residue betweentransmembrane segments 4 and 5 of the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows an alignment between Drosophila melanogaster andCaenorhabditis elegans MSC homologs.

DETAILED DESCRIPTION OF THE INVENTION INTRODUCTION

[0028] The present invention provides, for the first time, nucleic acidsencoding a eukaryotic mechanosensory transduction channel (MSC) protein.Mutations in these nucleic acids and the proteins they encode areresponsible for the “no-mechanoreceptor potential” phenotype inDrosophila, a phenotype involving uncoordination, often to the point oflethality, and a loss of mechanoreceptor potential in the bristles ofmutant flies (Kernan et al., Neuron 12:1195-1206 (1994)). The proteinsencoded by these nucleic acids form channels (e.g., as tetramers) thatcan directly detect mechanical stimuli and convert them into electricalsignals. These proteins can detect mechanical stimuli in any of a numberof sensory cells, such as neuronal sensory cells, hair cells, andothers. These nucleic acids and the proteins they encode can be used asprobes for sensory cells in animals, and can be used to diagnose andtreat any of a number of human conditions involving inherited, casual,or environmentally-induced loss of mechanosensory transduction activity.

[0029] The present invention also provides methods of screening formodulators, e.g., activators, inhibitors, enhancers, etc., ofmechanosensory transduction channels. Such modulators would be useful toalter mechanosensory transduction activity in an animal, e.g., for thetreatment of any of a number of human disorders. Thus, the inventionprovides assays for mechanosensory transduction modulation, where theMSC proteins act as a direct or indirect reporter for mechanosensorytransduction activity. MSC proteins can be used in assays, in vitro, invivo, or ex vivo, to detect changes in ion flux, ion concentration,membrane potential, signal transduction, transcription, or otherbiological or biophysical effects of mechanical stimulus detection.

[0030] In one embodiment, MSC proteins can be used as indirect reportersvia attachment to a second reporter molecule such as green fluorescentprotein (see, e.g., Mistili & Spector, Nature Biotechnology, 15:961-964(1997)). In one embodiment, MSC proteins are recombinantly expressed incells, e.g., Xenopus oocytes, and modulation of mechanosensorytransduction is assayed by detecting changes in transmembrane potential,mechanosensory potential, intracellular ion concentration, ion flux, andthe like.

[0031] In certain embodiments, potential modulators are identified byvirtue of an ability to physically interact with an MSC protein. Assaysfor physically-interacting molecules would provide an efficient primaryscreen for candidate MSC modulators, and, in addition, would allow theidentification of proteins and other compounds that naturally interactwith MSC proteins in vivo.

[0032] The invention also provides methods of detecting MSC nucleic acidand protein expression, allowing investigation into mechanosensoryregulation and the identification of mechanosensory cells. The presentnucleic acids and proteins can also be used to genotype an animal,including humans, for forensic, paternity, epidemiological, or otherinvestigations. The present invention also provides conserved sequencesfound in multiple MSC sequences, allowing the identification of evendistantly related MSC homologs (see, for example, SEQ ID NOs:7-9). Inaddition, the present invention provides methods for identifyingmutations in a mechanosensory transduction channel protein thateliminate or reduce function of the channel. Such mutations likelyunderlie one or more of the human conditions involving loss ofmechanosensation discussed herein. As such, the invention providesmethods of diagnosing mechanosensory transduction defects in animals.

[0033] Functionally, the MSC proteins form, within a cell membrane, achannel that directly detects mechanical stimuli and, in response to thestimuli, allows the influx of cations into a cell, thereby depolarizingthe cell and initiating an electrical, i.e. neural, signal.

[0034] Structurally, the nucleotide sequences of MSCs (see, e.g., SEQ IDNOs: 1, 3, and 5, representing the Drosophila genomic, Drosophila cDNA,and Caenorhabditis elegans genomic sequences, respectively) encodepolypeptides of from about 1619-1709 amino acids with a predictedmolecular weight of about 177 kDa (see, e.g., SEQ ID NOs:2, 4, and 6).The MSC genes typically contain about 19 exons, encoding a protein withabout 27 ankyrin repeats and from 6-11, typically about 8, transmembranedomains. Such proteins are weakly related to the TRP family ofepithelial cation channels. MSC homologs from other species typicallyshare at least about 70% identity over a region of at least about 25amino acids in length, preferably 50 to 100 amino acids in length.

[0035] The present invention provides nucleic acids comprising an MSCwherein the nucleic acid is less than 120, 90, 60, 30, 20, 10, or 7 kb.In addition, nucleic acids comprising MSCs are provided wherein the MSCpolynucleotide is isolated away from its genomic neighbors, i.e., thenucleic acid does not comprise any genes that are located within thesame genomic region as the MSC gene.

[0036] The present invention also provides polymorphic variants of theMSC depicted in SEQ ID NO:2: variant #1, in which an isoleucine residueis substituted for a leucine residue at amino acid position 6; variant#2, in which a glycine residue is substituted for an alanine residue atamino acid position 13; and variant #3, in which an arginine residue issubstituted for a lysine residue at amino acid position 22.

[0037] The present invention also provides polymorphic variants of theMSC depicted in SEQ ID NO:4: variant #1, in which an isoleucine residueis substituted for a leucine residue at amino acid position 24; variant#2, in which an alanine residue is substituted for a glycine residue atamino acid position 26; and variant #3, in which an aspartic acidresidue is substituted for a glutamic acid residue at amino acidposition 30.

[0038] The present invention also provides mutated MSC sequences thateliminate mechanosensory transduction activity in vivo. For example,mutations that prematurely truncate MSC proteins in the ankyrin repeatregion, or missense mutations that alter a cysteine residue betweentransmembrane segments four and five, e.g., a C to Y substitution, havebeen discovered that eliminate or severely reduce MSC activity. Suchmutations can be used, e.g., to detect defects in mechanosensation,specifically in mechanosensory transduction channels, in an animal suchas a human.

[0039] Specific regions of MSC may be used to identify polymorphicvariants, interspecies homologs, and alleles of MSC. Such identificationcan be made in vitro, e.g., under stringent hybridization conditions orby PCR (e.g., using primers encoding SEQ ID NOs 7-9) and sequencing, orby using the sequence information provided herein in a computer systemfor comparison with other nucleotide sequences. Typically,identification of polymorphic variants and alleles of MSC proteins ismade by comparing an amino acid sequence of about 25 amino acids ormore, e.g., 50-100 amino acids. Amino acid identify of approximately atleast about 70% or above, preferably 80%, most preferably 90-95% orabove typically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of MSC protein. Sequence comparison canbe performed using any of the sequence comparison algorithms discussedherein. Antibodies that specifically bind to MSC protein or a conservedregion thereof can also be used to identify alleles, interspecieshomologs, and polymorphic variants.

[0040] Polymorphic variants, interspecies homologs, and alleles of MSCproteins can be confirmed by examining mechanosensory cell-specificexpression of the putative MSC homolog. Typically, an MSC protein havinga sequence of SEQ ID NO:2, 4, or 6 can be used as a positive control incomparison to the putative homolog. Such putative homologs are expectedto retain the MSC structure described herein, i.e. intracellular domainwith multiple, e.g., 27, ankyrin repeats, and a transmembrane domaincontaining multiple, e.g, 8, transmembrane domains.

[0041] The present invention also provides promoters, enhancers, 5′- and3′-untranslated regions, and numerous other regulatory elements thatcontrol the transcription, translation, mRNA stability, mRNAlocalization, and other factors regulating MSC expression. For example,SEQ ID NO:1 provides genom ic DNA sequence including MSC coding sequenceas well as upstream and downstream regulatory sequences, includingpromoter sequences, etc. Promoters and other regulatory sequences can beidentified using standard methods well known to those of skill in theart, including by homology to well conserved regulatory elements such asthe TATA box or other elements, as taught, e.g., in Ausubel et al.,supra, or in Lewin, Genes IV (1990). Promoter, enhancer, and otherregulatory elements can also be determined functionally, e.g., by fusingspecific regions of SEQ ID NO:1 to a reporter gene and determining whichregions are sufficient for expression of the reporter gene, or bymutagenizing specific regions of SEQ ID NO:1 and thereby determiningwhich regions are required for expression. Such methods are well knownto those of skill in the art. Any of the present regulatory elements canbe used in isolation or together, and can be used to drive theexpression of an MSC protein, a marker protein, or any protein or RNAthat is desirably expressed in a cell or other expression system. Inpreferred embodiments, an MSC regulatory element is used to drive theexpression of a protein, e.g., an MSC or a heterologous polypeptide, ina tissue-specific manner, i.e., specifically in mechanosensory cells.

[0042] MSC nucleotide and amino acid sequences can also be used toconstruct models of mechanosensory transduction cell proteins in acomputer system. Such models can be used, e.g., to identify compoundsthat may interact with, activate, or inhibit MSC protein channels. Suchcompounds can then be used for various applications, such as to modulatemechanosensory transduction activity in vivo or to investigate thevarious roles of MSC in mechanosensory transduction in vivo.

[0043] The isolation of MSC protein also provides a means for assayingfor inhibitors and activators of mechanosensory transduction channels,as well as for molecules, e.g., proteins, that interact with MSCproteins in vitro or in vivo. Biologically active MSC protein channelsare useful for testing inhibitors and activators of MSC asmechanosensory transduction channels using in vivo and in vitroexpression, e.g., in oocytes, and measuring MSC expression,phosphorylation state, membrane potential, mechanosensory potential,intra- or extra-cellular ion concentration, ion flux, and the like.Molecules can also be screened for the ability to physically interactwith, e.g., bind to, MSC proteins, fragments thereof, or MSC nucleicacids, e.g., MSC promoter sequences, as shown in SEQ ID NO:1 and SEQ IDNO:3. Such interacting molecules can interact with any part of an MSC,e.g., the extracellular domain, transmembrane domain region, orintracellular domain, e.g., an ankyrin repeat. Such molecules may beinvolved in, or used to identify molecules capable of modulating, anyaspect of MSC activity, including channel formation, detection of amechanical stimulus, opening and/or closing of the channel, ionspecificity of the channel, adaptation of the channel, or any otherfunctional or physical aspect of the channel.

[0044] The present invention also provides assays, preferably highthroughput assays, to identify molecules that interact with and/ormodulate an MSC polypeptide. In numerous assays, a particular domain ofan MSC is used, e.g., an extracellular, transmembrane, or intracellulardomain. In numerous embodiments, an extracellular domain is bound to asolid substrate, and used, e.g., to isolate enhancers, inhibitors, orany molecule that can bind to and/or modulate the activity of anextracellular domain of an MSC polypeptide. In certain embodiments, adomain of an MSC polypeptide, e.g., an extracellular, transmembrane, orintracellular domain, is fused to a heterologous polypeptide, therebyforming a chimeric polypeptide. Such chimeric polypeptides are useful,e.g., in assays to identify modulators of an MSC polypeptide.

[0045] Such modulators and interacting molecules can be used for variouspurposes, such as to further investigate mechanosensory transductionchannel activity in animal cells, or to modulate mechanosensorytransduction activity in cells, e.g. to treat one or more conditionsassociated with a mechanosensory defect. It will be appreciated that inany of the binding assays or the in vitro or in vivo functional assaysdescribed herein, a full-length MSC can be used, or, alternatively, afragment of an MSC can be used, for example a region containing only theankyrin repeats, containing only the transmembrane domains, containingonly the extracellular domain, or containing only a fragment of anythese regions, will be used. Further, such fragments can be used alone,or fused to a heterologous protein any other molecule.

[0046] Definitions

[0047] The term “mechanosensory transduction protein” refers to apolypeptide that, when expressed in a cell or an oocyte, confers ontothe cell an ability to detect changes in pressure, motion, or any othermechanical stimulus as described herein. Such proteins can be expressednaturally or recombinantly, and can confer such activity on the cell invitro, in vivo, or ex vivo. Typically, such proteins will be at leastabout 70% identical to an amino acid sequence of SEQ ID NO:2, 4, or 6,and will include intracellular domains, including ankyrin repeats, andtransmembrane domains. However, such proteins can also refer to one ormore domains of these sequences in isolation, e.g., the ankyrin repeats,the extracellular domain, the transmembrane domains, or any subfragmentsthereof, alone. Such proteins can be involved in any mechanosensoryprocess, such as tactile sensation, proprioception, hearing,baroreception, and others.

[0048] The term “MSC protein” refers to polymorphic variants, alleles,mutants, and interspecies homologs that: (1) have about 70% amino acidsequence identity, preferably about 85-90% amino acid sequence identityto SEQ ID NOS:2, 4, or 6 over a window of about 25 amino acids,preferably 50-100 amino acids; (2) bind to antibodies raised against animmunogen comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, 4, 6-9, and conservatively modified variantsthereof; (3) specifically hybridize (with a size of at least about 500,preferably at least about 900 nucleotides) under stringent hybridizationand/or wash conditions to a sequence selected from the group consistingof SEQ ID NO:1, 3, and 5, and conservatively modified variants thereof;or (4) are amplified by primers that specifically hybridize understringent hybridization conditions to the same sequence as a degenerateprimer sets encoding SEQ ID NOS:7-9.

[0049] “Biological sample” as used herein is a sample of biologicaltissue or fluid that contains an MSC protein or nucleic acid encoding anMSC protein. Such samples include, but are not limited to, tissueisolated from humans, mice, rats, and other animals. Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. A biological sample is typically obtained from aeukaryotic organism, such as insects, protozoa, birds, fish, reptiles,and preferably a mammal such as rat, mouse, cow, dog, guinea pig, orrabbit, and most preferably a primate such as chimpanzees or humans.Preferred tissues include tissues involved in mechanosensation, such asthe inner ear or any mechanosensory epithelial or neural tissue.

[0050] The phrase “functional effects” in the context of assays fortesting compounds that modulate MSC protein-mediated mechanosensorytransduction includes the determination of any parameter that isindirectly or directly under the influence of the channel. It includeschanges in ion flux, membrane potential, current flow, transcription,MSC protein phosphorylation or dephosphorylation, signal transduction,in vitro, in vivo, and ex vivo and also includes other physiologiceffects such increases or decreases of neurotransmitter or hormonerelease.

[0051] By “determining the functional effect” is meant assays for acompound that increases or decreases a parameter that is indirectly ordirectly under the influence of MSC proteins. Such functional effectscan be measured by any means known to those skilled in the art, e.g.,patch clamping, voltage-sensitive dyes, whole-cell currents,radioisotope efflux, inducible markers, oocyte MSC expression; tissueculture cell MSC expression; transcriptional activation of MSC protein;ligand-binding assays; membrane potential and conductance changes;ion-flux assays; changes in intracellular calcium levels;neurotransmitter release, and the like.

[0052] A “physical effect” in the context of assays for testing theability of a compound to affect the activity of or bind to an MSCpolypeptide refers to any detectable alteration in the physical propertyor behavior of an MSC polypeptide due to an interaction with aheterologous compound, or any detection of a physical interaction using,e.g., electrophoretic, chromatographic, or immunologically-based assay,or using a two-hybrid screen as described infra. For example, a physicaleffect can include any alteration in any biophysical property of an MSCchannel comprising an MSC polypeptide, e.g., the cation specificity ormechanical sensitivity of the channel, or any structural or biochemicalproperties of an MSC polypeptide, e.g., its secondary, tertiary, orquaternary structure, hydrodynamic properties, spectral properties,chemical properties, or any other such property as described, e.g., inCreighton, Proteins (1984).

[0053] “Inhibitors,” “activators,” and “modulators” of MSC refer to anyinhibitory or activating molecules identified using in vitro and in vivoassays for mechanosensory transduction, e.g., agonists, antagonists, andtheir homologs and mimetics. Inhibitors are compounds that decrease,block, prevent, delay activation, inactivate, desensitize, or downregulate mechanosensory transduction, e.g., antagonists. Activators arecompounds that increase, open, activate, facilitate, enhance activation,sensitize or up-regulate mechanosensory transduction, e.g., agonists.Modulators include genetically-modified versions of MSC, e.g., withaltered activity, as well as naturally-occurring and synthetic ligands,antagonists, agonists, small chemical molecules and the like. Suchassays for inhibitors and activators include, e.g., expressing MSCprotein in cells or cell membranes, applying putative modulatorcompounds, and then determining the functional effects on mechanosensorytransduction, as described above. Samples or assays comprising MSC thatare treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of inhibition. Control samples(untreated with inhibitors) are assigned a relative MSC activity valueof 100%. Inhibition of MSC is achieved when the C activity valuerelative to the control is about 80%, preferably 50%, more preferably25-1%. Activation of MSCs is achieved when the MSC activity valuerelative to the control is 110%, more preferably 150%, more preferably200-500%, more preferably 1000-3000% higher.

[0054] “Biologically active” MSC refers to an MSC protein, or a nucleicacid encoding the MSC protein, having mechanosensory transductionactivity as described above, involved in mechanosensory transduction inmechanosensory cells.

[0055] The terms “isolated” “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis orhigh-performance liquid chromatography. A protein that is thepredominant species present in a preparation is substantially purified.In particular, an isolated MSC nucleic acid is separated, e.g., fromopen reading frames or fragments of open reading frames, e.g., thatnaturally flank the MSC gene and encode proteins other than MSC protein.An isolated MSC nucleic acid is typically contiguous, i.e., heterologoussequences are typically not embedded in the MSC nucleic acid sequence,although heterologous sequences are often found ajoining an isolated MSCnucleic acid sequence. The term “purified” denotes that a nucleic acidor protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

[0056] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0057] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, MRNA, oligonucleotide, andpolynucleotide.

[0058] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

[0059] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group., e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0060] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes (A, T, G, C, U, etc.).

[0061] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which theany position of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues to yield a codon encoding thesame amino acid residue (Batzer et al., Nucleic Acid Res. 19:5081(1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossoliniet al., Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy ofthe genetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon in an amino acid herein, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

[0062] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants and alleles of the invention.

[0063] The following groups each contain amino acids that areconservative substitutions for one another:

[0064] 1) Alanine (A), Glycine (G);

[0065] 2) Serine (S), Threonine (T);

[0066] 3) Aspartic acid (D), Glutamic acid (E);

[0067] 4) Asparagine (N), Glutarnine (Q);

[0068] 5) Cysteine (C), Methionine (M);

[0069] 6) Arginine (R), Lysine (K), Histidine (H);

[0070] 7) Isoleucine (I), Leucine (L), Valine (V); and

[0071] 8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,Creighton, Proteins (1984) for a discussion of amino acid properties).

[0072] A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins which can be madedetectable, e.g., by incorporating a radiolabel into the peptide or usedto detect antibodies specifically reactive with the peptide.

[0073] A “labeled nucleic acid probe or oligonucleotide” is one that isbound, either covalently, through a linker or a chemical bond, ornoncovalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe.

[0074] As used herein, a “nucleic acid probe or oligonucleotide” isdefined as a nucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

[0075] The term “recombinant” when used with reference, e.g., to a cell,or nucleic acid, protein, or vector, indicates that the cell, nucleicacid, protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

[0076] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0077] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

[0078] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0079] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Such sequencesare then said to be “substantially identical.” This definition alsorefers to the complement of a test sequence. Preferably, the percentidentity exists over a region of the sequence that is at least about 25amino acids in length, more preferably over a region that is 50 or 100amino acids in length.

[0080] For sequence comparison, one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

[0081] A “comparison window”, as used herein, includes reference to asegment of any one of the number of contiguous positions selected fromthe group consisting of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection.

[0082] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984).

[0083] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T, and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands.

[0084] The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

[0085] An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below.

[0086] The phrase “selectively (or specifically) hybridizes to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent hybridization conditionswhen that sequence is present in a complex mixture (e.g., total cellularor library DNA or RNA).

[0087] The phrase “stringent hybridization conditions,” or “stringentwash conditions,” refers to conditions under which a probe willhybridize to its target subsequence, typically in a complex mixture ofnucleic acid, but to no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization. Washes can beperformed for varying amounts of time, e.g., 5 minutes, 15 minutes, 30minutes, 1 hour or more. Exemplary stringent hybridization or washconditions can be as following: 50% formamide, 5×SSC, and 1% SDS,incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with washin 0.2×SSC, and 0.1% SDS at 65° C.

[0088] Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions,” or“moderately stringent wash conditions,” include a hybridization in abuffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSCat 45° C. Washes can be performed for varying amounts of time, e.g., 5minutes, 15 minutes, 30 minutes, 1 hour or more. A positivehybridization is at least twice background. Those of ordinary skill willreadily recognize that alternative hybridization and wash conditions canbe utilized to provide conditions of similar stringency.

[0089] A further indication that two polynucleotides are substantiallyidentical is if the reference sequence, amplified by a pair ofoligonucleotide primers, can then be used as a probe under stringenthybridization and/or wash conditions to isolate the test sequence from acDNA or genomic library, or to identify the test sequence in, e.g., anorthern or Southern blot. Alternatively, another indication that thesequences are substantially identical is if the same set of PCR primerscan be used to amplify both sequences.

[0090] “Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0091] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each air having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0092] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

[0093] A “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

[0094] An “anti-MSC” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the MSC gene, cDNA, or asubsequence thereof.

[0095] The term “immunoassay” is an assay that uses an antibody tospecifically bind an antigen. The immunoassay is characterized by theuse of specific binding properties of a particular antibody to isolate,target, and/or quantify the antigen.

[0096] The phrase “specifically (or selectively) binds” to an antibodyor “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.For example, polyclonal antibodies raised to MSC protein from specificspecies such as rat, mouse, or human can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withMSC and not with other proteins, except for polymorphic variants andalleles of MSC. This selection may be achieved by subtracting outantibodies that cross-react with MSC proteins from other species. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

[0097] The phrase “selectively associates with” refers to the ability ofa nucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

[0098] By “host cell” is meant a cell that contains an expression vectorand supports the replication or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells such as CHO, HeLaand the like, e.g., cultured cells, explants, and cells in vivo.

[0099] Isolation of MSC Nucleic Acids

[0100] General Recombinant DNA Methods

[0101] This invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

[0102] For nucleic acids, sizes are given in either kilobases (kb) orbase pairs (bp). These are estimates derived from agarose or acrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

[0103] Oligonucleotides that are not commercially available can bechemically synthesized according to the solid phase phosphoramiditetriester method first described by Beaucage & Caruthers, TetrahedronLetts. 22:1859-1862 (1981), using an automated synthesizer, as describedin Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson &Reanier, J. Chrom. 255:137-149 (1983).

[0104] The sequence of the cloned genes and synthetic oligonucleotidescan be verified after cloning using, e.g., the chain termination methodfor sequencing double-stranded templates of Wallace et al., Gene16:21-26 (1981).

[0105] Cloning MSC Nucleic Acids

[0106] In general, the nucleic acid sequences encoding MSC and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries by hybridization with a probe, or isolated using amplificationtechniques with oligonucleotide primers. For example, MSC sequences aretypically isolated from mammalian nucleic acid (genomic or cDNA)libraries by hybridizing with a nucleic acid probe, the sequence ofwhich can be derived from SEQ ID NOS:1, 3, or 5. MSC RNA and cDNA can beisolated from any of a number of tissues, such as hair cells of theinner ear, sensory neurons, or any other mechanosensory cell.

[0107] Amplification techniques using primers can also be used toamplify and isolate an MSC polynucleotide from DNA or RNA. Thedegenerate primers encoding the following amino acid sequences can alsobe used to amplify a sequence of MSC: SEQ ID NOS:7-9 (see, e.g.,Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)). Theseprimers can be used, e.g., to amplify either the full length sequence ora probe of one to several hundred nucleotides, which is then used toscreen a mammalian library for full-length MSC sequences.

[0108] Nucleic acids encoding MSC proteins can also be isolated fromexpression libraries using antibodies as probes. Such polyclonal ormonoclonal antibodies can be raised using polypeptides comprising thesequence of, e.g., SEQ ID NOS:2, 4, 6, 7, 8 or 9.

[0109] cDNA and Genomic Libraries

[0110] MSC polymorphic variants, alleles, and interspecies homologs thatare substantially identical to MSC proteins can be isolated using MSCnucleic acid probes, and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone MSC and MSC polymorphic variants, alleles, andinterspecies homologs, by detecting expressed homologs immunologicallywith antisera or purified antibodies made against MSC, which alsorecognize and selectively bind to the MSC homolog.

[0111] To make a cDNA library, one should choose a source that is richin MSC mRNA, e.g., inner ear tissue or other sources of mechanosensorycells, e.g., sensory epithelial cells or neurons. The mRNA is then madeinto cDNA using reverse transcriptase, ligated into a recombinantvector, and transfected into a recombinant host for propagation,screening and cloning. Methods for making and screening cDNA librariesare well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra).

[0112] For a genomic library, the DNA is extracted from the tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

[0113] Amplification Methods

[0114] An alternative method of isolating MSC nucleic acid and itshomologs combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of MSC directly from mRNA, from cDNA, from genomic librariesor cDNA libraries. Degenerate oligonucleotides can be designed toamplify MSC homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of MSC-encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

[0115] Gene expression of MSC protein can be analyzed by techniquesknown in the art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A⁺ RNA, Northern blotting, dot blotting,in situ hybridization, RNase protection, probing DNA microchip arrays,and the like. In one embodiment, high density oligonucleotide analysistechnology (e.g., GeneChip™) is used to identify homologs andpolymorphic variants of MSC. In the case where the homologs beingidentified are linked to a known disease, they can be used withGeneChip™ as a diagnostic tool in detecting the disease in a biologicalsample, see, e.g., Gunthand et al., AIDS Res. Hum. Retroviruses 14:869-876 (1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al.,Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat. Biotechnol.14:1675-1680 (1996); Gingeras et al., Genome Res. 8:435-448 (1998);Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

[0116] Synthetic oligonucleotides can be used to construct recombinantMSC genes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the MSC nucleic acid. The specificsubsequence is then ligated into an expression vector.

[0117] The nucleic acid encoding the MSC protein is typically clonedinto intermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors.

[0118] Expressing Nucleic Acids in Prokaryotes and Eukaryotes

[0119] Expression Vectors

[0120] To obtain high level expression of a cloned gene or nucleic acid,such as those cDNAs encoding an MSC protein, one typically subclones MSCinto an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the MSC protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

[0121] Promoters

[0122] The promoter used to direct expression of a heterologous nucleicacid depends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

[0123] In addition to the promoter, the expression vector typicallycontains a transcription unit or expression cassette that contains allthe additional elements required for the expression of the MSC-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding MSCprotein and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding MSC protein may typically be linked to acleavable signal peptide sequence to promote secretion of the encodedprotein by the transformed cell. Such signal peptides would include,among others, the signal peptides from tissue plasminogen activator,insulin, and neuron growth factor, and juvenile hormone esterase ofHeliothis virescens. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

[0124] Other Elements

[0125] In addition to a promoter sequence, the expression cassetteshould also contain a transcription termination region downstream of thestructural gene to provide for efficient termination. The terminationregion may be obtained from the same gene as the promoter sequence ormay be obtained from different genes.

[0126] The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

[0127] Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

[0128] Some expression systems have markers that provide geneamplification such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. Alternatively, high yield expressionsystems not involving gene amplification are also suitable, such asusing a baculovirus vector in insect cells, with a MSC encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

[0129] The elements that are typically included in expression vectorsalso include a replicon that functions in E. coli, a gene encodingantibiotic resistance to permit selection of bacteria that harborrecombinant plasmids, and unique restriction sites in nonessentialregions of the plasmid to allow insertion of eukaryotic sequences. Theparticular antibiotic resistance gene chosen is not critical, any of themany resistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

[0130] Transfection Methods

[0131] Standard transfection methods are used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofMSC protein, which are then purified using standard techniques (see,e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed., 1990)). Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

[0132] Any of the well known procedures for introducing foreignnucleotide sequences into host cells may be used. These include the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, liposomes, microinjection, plasma vectors, viralvectors and any of the other well known methods for introducing clonedgenomic DNA, cDNA, synthetic DNA or other foreign genetic material intoa host cell (see, e.g., Sambrook et al., supra). It is only necessarythat the particular genetic engineering procedure used be capable ofsuccessfully introducing at least one gene into the host cell capable ofexpressing MSC.

[0133] After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofMSC, which is recovered from the culture using standard techniquesidentified below.

[0134] Purification of MSC Proteins

[0135] Either naturally occurring or recombinant MSC protein can bepurified for use in functional assays. Preferably, recombinant MSC ispurified. Naturally occurring MSC is purified, e.g., from mammaliantissue such as inner ear tissue or other tissues includingmechanosensory cells. Recombinant MSC is purified from any suitableexpression system.

[0136] MSC protein may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

[0137] A number of procedures can be employed when recombinant MSC isbeing purified. For example, proteins having established molecularadhesion properties can be reversibly fused to MSC. With the appropriateligand, MSC can be selectively adsorbed to a purification column andthen freed from the column in a relatively pure form. The fused proteinis then removed by enzymatic activity. Finally MSC could be purifiedusing immunoaffinity columns.

[0138] Purification from Recombinant Bacteria

[0139] Recombinant proteins are expressed by transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

[0140] Proteins expressed in bacteria may form insoluble aggregates(“inclusion bodies”). Several protocols are suitable for purification ofMSC inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

[0141] If necessary, the inclusion bodies are solubilized, and the lysedcell suspension is typically centrifuged to remove unwanted insolublematter. Proteins that formed the inclusion bodies may be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein. Other suitable buffers are known to those skilled in the art.MSC is separated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

[0142] Alternatively, it is possible to purify MSC protein from bacteriaperiplasm. After lysis of the bacteria, when MSC is exported into theperiplasm of the bacteria, the periplasmic fraction of the bacteria canbe isolated by cold osmotic shock in addition to other methods known toskill in the art. To isolate recombinant proteins from the periplasm,the bacterial cells are centrifuged to form a pellet. The pellet isresuspended in a buffer containing 20% sucrose. To lyse the cells, thebacteria are centrifuged and the pellet is resuspended in ice-cold 5 mMMgSO₄ and kept in an ice bath for approximately 10 minutes. The cellsuspension is centrifuged and the supernatant decanted and saved. Therecombinant proteins present in the supernatant can be separated fromthe host proteins by standard separation techniques well known to thoseof skill in the art.

[0143] Standard Protein Purification Techniques

[0144] Solubility Fractionation

[0145] Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

[0146] Size Differential Filtration

[0147] The molecular weight of MSC protein can be used to isolated itfrom proteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

[0148] Column Chromatography

[0149] MSC proteins can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

[0150] Affinity-Based Techniques

[0151] Any of a number of affinity based techniques can be used toisolate MSC proteins from cells, cell extracts, or other sources. Forexample, affinity columns can be made using anti-MSC antibodies or otherMSC-binding proteins, or physically-interacting proteins can beidentified by co-immunoprecipitation or other methods. Such methods arewell known to those of skill in the art and are taught, e.g., in Ausubelet al., Sambrook et al., Harlow and Lane, all supra.

[0152] Immunological Detection

[0153] In addition to the detection of MS genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect MSC proteins, e.g., to identify mechanosensorycells and variants of MSC proteins. Immunoassays can be used toqualitatively or quantitatively analyze MSC proteins. A general overviewof the applicable technology can be found in Harlow & Lane, Antibodies:A Laboratory Manual (1988).

[0154] Antibodies to MSC Proteins

[0155] Methods of producing polyclonal and monoclonal antibodies thatreact specifically with MSC proteins are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

[0156] A number of MSC peptides or a full-length protein may be used toproduce antibodies specifically reactive with MSC protein. For example,recombinant MSC protein, or an antigenic fragment thereof, is isolatedas described herein. Recombinant protein can be expressed in eukaryoticor prokaryotic cells as described above, and purified as generallydescribed above. Recombinant protein is the preferred immunogen for theproduction of monoclonal or polyclonal antibodies. Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used as an immunogen. Naturallyoccurring protein may also be used either in pure or impure form. Theproduct is then injected into an animal capable of producing antibodies.Either monoclonal or polyclonal antibodies may be generated, forsubsequent use in immunoassays to measure the protein.

[0157] Methods of production of polyclonal antibodies are known to thoseof skill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to MSC proteins. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see, Harlow & Lane, supra).

[0158] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989).

[0159] Monoclonal antibodies and polyclonal sera are collected andtitered against the immunogen protein in an immunoassay, for example, asolid phase immunoassay with the immunogen immobilized on a solidsupport. Typically, polyclonal antisera with a titer of 10⁴ or greaterare selected and tested for their cross reactivity against non-MSCproteins or even other related proteins from other organisms, using acompetitive binding immunoassay. Specific polyclonal antisera andmonoclonal antibodies will usually bind with a K_(d) of at least about0.1 mM, more usually at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better.

[0160] Once MSC specific antibodies are available, MSC proteins can bedetected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

[0161] Immunological Binding Assays

[0162] MSC proteins can be detected and/or quantified using any of anumber of well recognized immunological binding assays (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunologicalbinding assays (or immunoassays) typically use an antibody thatspecifically binds to a protein or antigen of choice (in this case theMSC protein or antigenic subsequence thereof). The antibody (e.g.,anti-MSC) may be produced by any of a number of means well known tothose of skill in the art and as described above.

[0163] Immunoassays also often use a labeling agent to specifically bindto and label the complex formed by the antibody and antigen. Thelabeling agent may itself be one of the moieties comprising theantibody/antigen complex. Thus, the labeling agent may be a labeled MSCpolypeptide or a labeled anti-MSC antibody. Alternatively, the labelingagent may be a third moiety, such a secondary antibody, thatspecifically binds to the antibody/MSC complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see, e.g., Kronval et al., J. Immunol. 111:1401-1406(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). Thelabeling agent can be modified with a detectable moiety, such as biotin,to which another molecule can specifically bind, such as streptavidin. Avariety of detectable moieties are well known to those skilled in theart.

[0164] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, antigen, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0165] Non-Competitive Formats

[0166] Immunoassays for detecting MSC proteins in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-MSC antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture MSC proteins present in the testsample. The MSC protein is thus immobilized and then bound by a labelingagent, such as a second MSC antibody bearing a label. Alternatively, thesecond antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second or third antibody istypically modified with a detectable moiety, such as biotin, to whichanother molecule specifically binds, e.g., streptavidin, to provide adetectable moiety.

[0167] Competitive Formats

[0168] In competitive assays, the amount of MSC proteins present in thesample is measured indirectly by measuring the amount of a known, added(exogenous) MSC proteins displaced (competed away) from an anti-MSCantibody by the unknown MSC protein present in a sample. In onecompetitive assay, a known amount of MSC protein is added to a sampleand the sample is then contacted with an antibody that specificallybinds to MSC proteins. The amount of exogenous MSC protein bound to theantibody is inversely proportional to the concentration of MSC proteinpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of MSC proteinbound to the antibody may be determined either by measuring the amountof MSC protein present in a MSC protein/antibody complex, oralternatively by measuring the amount of remaining uncomplexed protein.The amount of MSC protein may be detected by providing a labeled MSCprotein molecule.

[0169] A hapten inhibition assay is another preferred competitive assay.In this assay the known MSC protein, is immobilized on a solidsubstrate. A known amount of anti-MSC antibody is added to the sample,and the sample is then contacted with the immobilized MSC protein. Theamount of anti-MSC antibody bound to the known immobilized MSC proteinis inversely proportional to the amount of MSC protein present in thesample. Again, the amount of immobilized antibody may be detected bydetecting either the immobilized fraction of antibody or the fraction ofthe antibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

[0170] Cross-Reactivity Determination

[0171] Immunoassays in the competitive binding format can also be usedfor crossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NOS:1, 3, or 5 can be immobilized to a solidsupport. Proteins (e.g., MSC proteins and homologs) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability of MSCprotein encoded by SEQ ID NO:1, 3, or 5 to compete with itself. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith each of the added proteins listed above are selected and pooled.The cross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs. In one embodiment, antibodies thatcrossreact with MSC proteins from a different species are selectivelyremoved, thereby enhancing the species-specificity of the antisera. Forexample, to obtain antibodies that specifically react with DrosophilaMSC, the ability of SEQ ID NO:4 and SEQ ID NO:6 to compete for bindingto antisera directed against SEQ ID NO:4 are compared, and antibodiesthat cross-react with SEQ ID NO:6 selectively removed.

[0172] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein, thought to be perhaps an allele or polymorphic variant of MSCprotein, to the immunogen protein (i.e., MSC protein of SEQ ID NOS:2, 4,6-9). In order to make this comparison, the two proteins are eachassayed at a wide range of concentrations and the amount of each proteinrequired to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired to inhibit 50% of binding is less than 10 times the amount ofthe protein encoded by SEQ ID NOS:1, 3, or 5 that is required to inhibit50% of binding, then the second protein is said to specifically bind tothe polyclonal antibodies generated to an MSC protein immunogen.

[0173] Other Formats

[0174] Western blot (immunoblot) analysis is used to detect and quantifythe presence of MSC protein in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind MSC protein. The anti-MSC antibodies specificallybind to the MSC protein on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-MSC antibodies.

[0175] Other assay formats include liposome immunoassays (LIA), whichuse liposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,Amer. Clin. Prod. Rev. 5:34-41 (1986)).

[0176] Reduction of Non-Specific Binding

[0177] One of skill in the art will appreciate that it is oftendesirable to minimize non-specific binding in immunoassays. Particularlywhere the assay involves an antigen or antibody immobilized on a solidsubstrate, it is desirable to minimize the amount of non-specificbinding to the substrate. Means of reducing such non-specific bindingare well known to those of skill in the art. Typically, this techniqueinvolves coating the substrate with a proteinaceous composition. Inparticular, protein compositions such as bovine serum albumin (BSA),nonfat powdered milk, and gelatin are widely used, with powdered milkbeing most preferred.

[0178] Labels

[0179] The particular label or detectable group used in the assay is nota critical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

[0180] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

[0181] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to another molecule (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize MSC protein, orsecondary antibodies that recognize anti-MSC protein.

[0182] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, or oxidotases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

[0183] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0184] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0185] Assays for Modulators of Mechanosensory Transduction

[0186] In numerous embodiments of this invention, assays will beperformed to detect compounds that affect mechanosensory transduction ina cell. Such assays can involve the identification of compounds thatinteract with MSC proteins, either physically or genetically, and canthus rely on any of a number of standard methods to detect physical orgenetic interactions between compounds. Such assays can also involve thedetection of mechanosensory transduction in a cell or cell membrane,either in vitro or in vivo, and can thus involve the detection oftransduction activity in the cell through any standard assay, e.g., bymeasuring ion flux, changes in membrane potential, and the like. Suchcell-based assays can be performed in any type of cell, e.g., a sensorycell that naturally expresses MSC, a cultured cell that produces MSC dueto recombinant expression, or, preferably, an oocyte that is induced toproduce MSC through any of a number of means, as described infra.

[0187] In any of the binding or functional assays described herein, invivo or in vitro, any MSC protein, or any derivative, variation,homolog, or fragment of an MSC protein, can be used. Preferably, the MSCprotein is at least about 70% identical to SEQ ID NO:2, 4, or 6, and/orcomprises SEQ ID NO:7, 8, or 9. In numerous embodiments, a fragment ofan MSC protein is used. For example, a fragment that contains only theextracellular region, the ankyrin repeat region, or the transmembranedomains, i.e. the channel region (see, e.g., SEQ ID NOs:10-17), can beused. Such fragments can be used alone, in combination with other MSCfragments, or in combination with sequences from a heterologous protein,e.g., the fragments can be fused to a heterologous polypeptide, therebyforming a chimeric polypeptide. Any individual domain or sequence,however small, can readily be used in the present invention, e.g., asingle ankyrin repeat, transmembrane domain, etc., alone or incombination with other domains or with sequences from heterologousproteins. Such fragments and isolated domains of MSC proteins comprisean essential aspect of the present invention, and are of substantialimportance in the assays described herein.

[0188] Assays for MSC-Interacting Compounds

[0189] In certain embodiments, assays will be performed to identifymolecules that physically or genetically interact with MSC proteins.Such molecules can be any type of molecule, including polypeptides,polynucleotides, amino acids, nucleotides, carbohydrates, lipids, or anyother organic or inorganic molecule. Such molecules may representmolecules that normally interact with MSC to effect mechanosensation insensory cells, or may be synthetic or other molecules that are capableof interacting with MSC and which can potentially be used to modulateMSC activity in cells, or used as lead compounds to identify classes ofmolecules that can interact with and/or modulate MSC. Such assays mayrepresent physical binding assays, such as affinity chromatography,immunoprecipitation, two-hybrid screens, or other binding assays, or mayrepresent genetic assays as described infra.

[0190] Such interacting molecules may interact with any part of an MSCprotein, e.g., the extracellular domain, the transmembrane domainregion, or the intracellular domain, including the ankyrin repeats. MSCproteins act in sensory cells to depolarize the cell in response to amechanical input outside of the cell. As such, interacting molecules mayinclude those that interact with the extracellular domain of theprotein, and which may enhance, inhibit, or otherwise modulate thedetection of a mechanical input, and which may be part of, or interactwith, an extracellular structure involved in mechanical detection, suchas the stereocilium of a hair cell. An interacting molecule may alsointeract with the transmembrane domain region of the protein, and may beinvolved in, or capable of modulating, the formation of a channel, theopening or closing of a channel, etc. In addition, an interactingmolecule may interact with an intracellular part of a channel, e.g., anankyrin repeat, and be involved in, e.g., the function, regulation,adaptation, or any other aspect of channel activity.

[0191] The MSC protein used in such assays can be a full-length MSCprotein or any subdomain of an MSC protein. In preferred embodiments, afragment of an MSC protein comprising an extracellular domain of an MSCwill be used. Molecules that bind to the extracellular domain of an MSCare particularly useful for the identification of modulators of MSCactivity, as they are typically soluble and readily included in highthroughput screening assay formats, as described infra.

[0192] Assays for Physical Interactions

[0193] Compounds that interact with MSC proteins can be isolated basedon an ability to specifically bind to an MSC protein or fragmentthereof. In numerous embodiments, the MSC protein or protein fragmentwill be attached to a solid support. In one embodiment, affinity columnsare made using the MSC polypeptide, and physically-interacting moleculesare identified. It will be apparent to one of skill that chromatographictechniques can be performed at any scale and using equipment from manydifferent manufacturers (e.g., Pharmacia Biotech). In addition,molecules that interact with MSC proteins in vivo can be identified byco-immunoprecipitation or other methods, i.e. immunoprecipitating MSCproteins using anti-MSC antibodies from a cell or cell extract, andidentifying compounds, e.g., proteins, that are precipitated along withthe MSC protein. Such methods are well known to those of skill in theart and are taught, e.g., in Ausubel et al., Sambrook et al., Harlow &Lane, all supra.

[0194] Two-hybrid screens can also be used to identify polypeptides thatinteract in vivo with an MSC or a fragment thereof (Fields et al.,Nature 340:245-246 (1989)). Such screens comprise two discrete, modulardomains of a transcription factor protein, e.g., a DNA binding domainand a transcriptional activation domain, which are produced in a cell astwo separate polypeptides, each of which also comprises one of twopotentially binding polypeptides. If the two potentially bindingpolypeptides in fact interact in vivo, then the DNA binding and thetranscriptional activating domain of the transcription factor areunited, thereby producing expression of a target gene in the cell. Thetarget gene typically encodes an easily detectable gene product, e.g.,β-galactosidase, which can be detected using standard methods. In thepresent invention, an MSC polypeptide is fused to one of the two domainsof the transcription factor, and the potential MSC-binding polypeptides(e.g., encoded by a cDNA library) are fused to the other domain. Suchmethods are well known to those of skill in the art, and are taught,e.g., in Ausubel et al., supra.

[0195] Assays for Genetic Interactions

[0196] It is expected that MSCs are assembled into multi-proteincomplexes in which the interactions are mediated by the large number ofankyrin repeats found in the N terminus of the protein. Genetic screenscan thus be performed to identify such additional proteins that areinvolved in the transduction pathway. For example, genetic strains areproduced that possess only a partially functional nompC (MSC) gene,which confers an incomplete mechanical sensitivity to the fly. Ideally,a vial of these flies would produce only 10-20 viable homozygotes. Inthis sensitized genetic background, flies will be screened for mutationsin other genes that either suppress or enhance the survival of themutant flies. Flies will be mutagenized using any standard chemical,radiation-based, or genetic method and then crossed into theabove-described sensitized genetic background, followed by counting thenumber of homozygous progeny. Mutations that produce more than 10-20flies per vial are considered suppressors of nompC, and those thatproduce fewer flies are considered enhancers. Similar screens can beperformed using MSC genes in genetically tractable mammals, e.g., mice.

[0197] Assays for MSC Activity

[0198] The activity of MSC polypeptides, and any homolog, variant,derivative, or fragment thereof can be assessed using a variety of invitro and in vivo assays for mechanoreceptor potential, e.g., measuringcurrent, measuring membrane potential, measuring ion flux, e.g.,potassium or calcium, measuring transcription levels, measuringneurotransmitter levels, using e.g., voltage-sensitive dyes, radioactivetracers, patch-clamp electrophysiology, transcription assays, and thelike. Furthermore, such assays can be used to test for modulators, e.g.,inhibitors or activators, of MSC. Such modulators can be a protein,amino acid, nucleic acid, nucleotide, lipid, carbohydrate, or any typeof organic or inorganic molecule, including genetically altered versionsof MSC proteins. Such assays can be performed using any of a largenumber of cells, including oocytes, cultured cells, sensory epithelialor neural cells, and others, and can be present in vitro or in vivo.Such cells can contain naturally expressed MSC, can be induced toexpress MSC using recombinant or other methods, or can comprise MSC bydirect addition of the protein to the cell or cell membrane. In numerousembodiments, the cell or cell membrane comprising the MSC polypeptidewill be anchored to a solid support.

[0199] Preferably, the MSC proteins used in the assay is selected from apolypeptide having a sequence of SEQ ID NOS:2, 4, or 6, or aconservatively modified variant thereof. Alternatively, the MSC proteinused in the assay will be derived from a eukaryote and include an aminoacid subsequence having amino acid sequence identity SEQ ID NOS:2, 4, or6. Generally, the amino acid sequence identity will be at least 70%,preferably at least 85%, most preferably at least 90-95%. In preferredembodiments, a polypeptide comprising an extracellular domain is used,e.g., an extracellular domain of SEQ ID NO:2, 4, or 6. In suchembodiments, the extracellular domain is often fused to a heterologouspolypeptide, forming a chimeric polypeptide. Typically, such chimericpolypeptides will comprise an extracellular domain as well as multipletransmembrane domains, and will have mechanosensory transductionactivity.

[0200] Detecting Mechanosensory Transduction

[0201] In numerous embodiments of the present invention, assays will beperformed to detect alterations in an MSC protein, e.g., one expressedin a cell or cell membrane, or in mechanosensory transduction, ormechanoreceptor potential, in a cell or cell membrane, e.g., as a resultof a mutation in an MSC or due to the presence of an MSC-modulatingcompound. Mechanosensory transduction or mechanoreceptor potential canbe detected in any of a number of ways, including by detecting changesin ion flux, changes in polarization of a cell or cell membrane, changesin current, and other methods, including by measuring downstreamcellular effects, e.g., neuronal signaling.

[0202] Changes in ion flux may be assessed by determining changes inpolarization (i.e., electrical potential) of the cell or membraneexpressing MSC. One means to determine changes in cellular polarizationis by measuring changes in current (thereby measuring changes inpolarization) with voltage-clamp and patch-clamp techniques, e.g., the“cell-attached” mode, the “inside-out” mode, and the “whole cell” mode(see, e.g., Ackerman et al., New Engl. J. Med. 336:1575-1595 (1997)).Whole cell currents are conveniently determined using the standardmethodology (see, e.g., Hamil et al., PFlugers. Archiv. 391:85 (1981).Other known assays include: radioactive ion flux assays and fluorescenceassays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind etal., J. Membrane Biol. 88:67-75 (1988); Gonzales & Tsien, Chem. Biol.4:269-277 (1997); Daniel et al., J. Pharmacol. Meth. 25:185-193 (1991);Holevinsky et al., J. Membrane Biology 137:59-70 (1994)). Generally,candidate compounds are tested in the range from 1 pM to 100 mM.

[0203] The effects of the test compounds, or sequence variation, uponthe function of the MSC polypeptides can be measured by examining any ofthe parameters described above. In addition, any suitable physiologicalchange that affects MSC activity, or reflects MSC activity, can be usedto assess the influence of a test compound or sequence alteration on theMSC polypeptides of this invention. When the functional consequences aredetermined using intact cells or animals, one can also measure a varietyof effects such as transmitter release, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,northern blots), changes in cell metabolism such as cell growth or pHchanges, and other effects.

[0204] Preferred assays for mechanosensory transduction channels includecells, e.g., oocytes, that are loaded with ion or voltage sensitive dyesto report receptor activity. Assays for determining activity of suchreceptors can also use known agonists and antagonists for other cationchannels as negative or positive controls to assess activity of testedcompounds. In assays for identifying modulatory compounds (e.g.,agonists, antagonists), changes in the level of ions in the cytoplasm ormembrane voltage will be monitored using an ion-sensitive or membranevoltage fluorescent indicator, respectively. Among the ion-sensitiveindicators and voltage probes that may be employed are those disclosedin the Molecular Probes 1997 Catalog. In addition, changes incytoplasmic calcium, potassium, or other ion levels can be used toassess MSC function.

[0205] In vivo Assays

[0206] In certain embodiments, the mechanosensory activity of a cellwill be examined in vivo. Such embodiments are useful for, e.g.,examining the activity of an MSC or an MSC mutant, derivative, homolog,fragment, etc. Also, such assays are useful for detecting the activityof candidate MSC modulator in vivo. Potential MSCs can be produced intransgenic flies carrying the candidate cDNA driven by a suitable, e.g.a nompc, promoter/enhancer construct. These candidate channels can beexpressed in mechanosensory neurons of flies and their mechanoelectricalactivity measured with bristle recordings. Methods of producingtransgenic flies and methods of detecting mechanosensory transductionactivity in fly mechanosensory neurons are well known to those of skillin the art and are described, e.g., in Drosophila, a Practical Approach(Roberts, ed. 1986)), and in Keman et al. (1994), respectively.

[0207] Alternatively, it is possible to screen for molecules that canmimic NOMPC activity by performing the screen in a nompC mutantbackground. Those molecules that rescue the mutant phenotype can beconsidered potential MSCs.

[0208] Assays using Oocytes or Cultured Cells in vitro

[0209] Xenopus Oocytes

[0210] In preferred embodiments, MSC proteins are expressed in oocytesof the frog Xenopus laevis, and the mechanosensory transduction of theoocyte measured. Such assays are useful, e.g., to measure the activityof homologs, variants, derivatives, and fragments of MSC proteins, aswell as to measure the effect of candidate modulators on the activity ofMSC protein channels in the oocytes. In such embodiments, mRNA encodingthe MSC protein, or candidate MSC protein, is typically microinjectedinto the oocyte where it is translated. The MSC protein, and in somecases the candidate MSC, then forms a functional mechanosensorytransduction channel in the oocyte which can be studied using themethods described herein. In such embodiments, MSC cDNAs are typicallysubcloned into specialized transcription vectors in which the cDNAinsert is flanked by Xenopus hemoglobin 5′ and 3′ untranslated regions.Transcripts are made from both the sense and antisense strand of theplasmid and then polyadenylated using standard techniques. Thesetranscripts are then microinjected into Xenopus laevis oocytes. Afterallowing a sufficient time for translation, the oocytes are subjected tovoltage-clamp recording. Cell-attached patches of oocyte membrane areassayed for the presence of conductances provoked by the application ofmechanical force to the membrane, e.g., using small, calibrated pressureand vacuum steps applied through the patch pipette. Because Xenopusoocyte membranes contain an endogenous mechanically gated conductance,which is typically observed using these methods, the conductance due tothe heterologous MSC channel represents any additional conductance,i.e., beyond the background level, seen during a mechanical stimulus. Insuch assays, it is important to compare the sense- to the antisense- andmock-injected controls for the presence of mechanically gatedconductances.

[0211] Cultured Cells

[0212] In certain embodiments, MSC proteins are expressed in culturedcells, e.g., mammalian cells, and the mechanosensory transductionactivity of the cell determined. In such assays, cDNAs encoding known orcandidate MSC proteins are typically subcloned into commerciallyavailable cell expression vectors, e.g., mammalian cell expressionvectors, and then transfected into cultured cells. Expression vectors,transfection, and maintenance of animal cells are well known to those ofskill and are taught, e.g., in Ausubel et al., supra, and Freshney, TheCulture of Animal Cells (1993).

[0213] Cultured animal cells expressing MSC proteins, like theabove-described oocytes, are subjected to cell-attached patchvoltage-clamp recording during the application of mechanical stimulisuch as small, calibrated pressure and suction stimuli to the patch.Osmotic membrane stress can also serve as a mechanical stimulus. Again,as eukaryotic cells generally contain endogenous mechanically gated ionchannels, it is important to compare the transduction levels in thetransfected cells to those in the mock-transfected controls. Anymechanically-gated conductance detectable above the level of theendogenous conductance is due to the candidate channel.

[0214] Alternatively, because MSC channels conduct calcium ions,transfected cells are loaded with a fluorescent Ca²⁺ indicator dye andthen stimulated with hypo- and hyper-osmotic solutions while monitoringthe cell's fluorescence. Hyper- and hypo-osmotic solutions createmembrane stresses that open mechanically gated ion channels. In suchassays, the influx of Ca²⁺ causes an increase in fluorescence of theCa²⁺ indicator dye. As with the voltage-clamp recording, it is importantto compare the transfected and mock-transfected controls. Any increasedfluorescence in the transfected cells during the stimuli compared tothat observed in mock transfected cells is due to the presence of theMSC channel.

[0215] Biophysical Properties of MSC channels

[0216] The effect of a sequence alteration in an MSC channel, or of acandidate modulator on a channel, can also be assessed by examining theeffect of the sequence alteration or the compound on one or morestructural or biophysical properties that are typical of MSC channels.For example, MSC channels show very little voltage dependence, and areinstead gated by mechanical stimuli. Further, MSC channels have anon-specific cationic preference, i.e., they conduct many differentcations, including some large organic cations like tetramethyl ammoniumion (although weakly). The solution bathing these channels in theDrosophila bristle and in vertebrate hearing organs has a high potassiumion concentration (over 100 mM), which is very unusual for anextracellular fluid. Because of this, the principal current-carrying ionin vivo is K⁺, with a small portion of the current carried by Ca²⁺. Inaddition, as MSC channels are completely blocked in vivo by tetraethylammonium ions, it is expected that the channels are also refractory totetraethyl ammonium ions in heterologous systems. Further, MSC proteinsare in general refractory to Gd³⁺ ions, albeit at millimolarconcentrations; in our bristle recording system, however, flymechanoreceptor neurons are unaffected by Gd³⁺ treatment.

[0217] It will be appreciated that any of these characteristics, whichare typical of mechanosensory transduction channels in vivo, can beassessed in cell-attached patches in either oocytes or cultured cells toassess the effect of any potential modulator, mutation, or treatmentupon an MSC protein.

[0218] Candidate Modulators and MSC-binding Compounds

[0219] Using the present methods, any protein, amino acid, nucleic acid,nucleotide, carbohydrate, lipid, or any other organic or inorganicmolecule can be assessed for its ability to bind to or modulate theactivity of an MSC polypeptide. Such candidate modulators or bindingproteins can be deliberately designed, e.g., a putativedominant-negative form of an MSC polypeptide or a compound predicted tobind based on a computer-based structural analysis of the protein, orcan be identified using high efficiency assays to rapidly screen a largenumber of potential compounds, e.g., from a library of nucleic acids ora combinatorial peptide or chemical library.

[0220] Proteins

[0221] Any of a number of polypeptides can be used in the present assaysto determine their ability to bind to or modulate mechanosensorytransduction activity in an MSC-protein expressing cell. Suchpolypeptides can represent, e.g., a candidate protein or collection ofproteins encoded by a library of nucleic acids, can represent a putativedominant negative form or other variant of an MSC polypeptide, canrepresent a collection of peptide sequences, e.g., from a combinatorialpeptide library, or can be predicted using a computer-based structuralanalysis program.

[0222] Heterologous Proteins

[0223] Polypeptide modulators of MSC proteins can be identified using afluorescence-based screening strategy. In such approaches, cells arefirst induced to stably express an MSC protein, and then transfectedwith a cDNA clone of interest, e.g., representing adeliberately-selected candidate modulator or a collection of randomclones such as a cDNA library isolated from a sensory tissue. Thetransfected cells are then loaded with fluorescent Ca²⁺-indicator dyesand subjected to an osmotic stimulus or a mild mechanical treatment.Heterologous proteins that exert a modulatory effect on the MSC channelwill cause the cell to exhibit either an increase or a decrease in thefluorescence during the stimulus compared to a cell expressing the MSCprotein alone.

[0224] MSC Protein Fragments. e.g. Dominant Negative Forms

[0225] Because MSCs are thought be part of a multi-protein complex invivo, it is expected that a dominant-negative form of MSC can beproduced by designing an MSC that lacks mechanosensory transductionactivity but which can nevertheless interact in vivo with othermolecules involved in mechanosensory transduction. A “dominant-negative”MSC refers to any MSC whose presence reduces mechanosensory activity invivo, even in the presence of fully functional MSC protein. For example,overexpression of the ankyrin repeats alone (which are thought tofacilitate protein-protein interactions), or in combination with adefective channel domain, will likely lead to the disruption ofmechanical signaling. Alternatively, if these channels are comprised ofseveral homomeric subunits (e.g., single MSC polypeptide units),expression of the channel moiety alone will reduce mechanosensorysignaling in a dominant fashion.

[0226] In addition, because MSCs are weakly similar at a structurallevel to many voltage-activated channels, they could potentially containan endogenous “ball and chain” inactivator of the channel (see, e.g.,Antz et al., Nat Struct Biol 6(2):146-50 (1999)). Accordingly, one canpotentially identify such endogenous modulators by producing smallfragments of MSC, e.g., using a bacterial expression system, andassaying their ability to inhibit MSC protein activity in an assay asdiscussed supra.

[0227] Small Molecules

[0228] In numerous embodiments of this invention, test compounds will besmall chemical molecules or peptides. Essentially any chemical compoundcan be used as a potential modulator or ligand in the assays of theinvention, although most often compounds that can be dissolved inaqueous or organic (especially DMSO-based) solutions are used. Theassays are designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source toassays, which are typically run in parallel (e.g., in microtiter formatson microtiter plates in robotic assays). It will be appreciated thatthere are many suppliers of chemical compounds, including Sigma (St.Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.),Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

[0229] Combinatorial Libraries

[0230] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0231] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0232] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

[0233] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0234] High Throughput Screening

[0235] In one embodiment, the invention provides solid phase based invitro assays in a high throughput format, where the cell, cell membrane,or tissue comprising the MSC protein is attached to a solid phasesubstrate. In the high throughput assays of the invention, it ispossible to screen up to several thousand different modulators orligands in a single day. In particular, each well of a microtiter platecan be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100-about 1500 different compounds. It is possible to assayseveral different plates per day; assay screens for up to about6,000-20,000 different compounds is possible using the integratedsystems of the invention. More recently, microfluidic approaches toreagent manipulation have been developed.

[0236] Computer-Based Assays

[0237] Yet another assay for compounds that modulate MSC activityinvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of MSC proteins based onthe structural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind heterologous molecules. Theseregions are then used to identify molecules that bind to the protein.

[0238] The three-dimensional structural model of the protein isgenerated by entering protein amino acid sequences of at least 10 aminoacid residues or corresponding nucleic acid sequences encoding a MSCpolypeptide into the computer system. For example, the amino acidsequence of the polypeptide is selected from the group consisting of SEQID NOS:2, 4, and 6, and conservatively modified versions thereof. Theamino acid sequence represents the primary sequence or subsequence ofthe protein, which encodes the structural information of the protein. Atleast 10 residues of the amino acid sequence (or a nucleotide sequenceencoding 10 amino acids) are entered into the computer system fromcomputer keyboards, computer readable substrates that include, but arenot limited to, electronic storage media (e.g., magnetic diskettes,tapes, cartridges, and chips), optical media (e.g., CD-ROM), informationdistributed by internet sites, and by RAM. The three-dimensionalstructural model of the protein is then generated by the interaction ofthe amino acid sequence and the computer system, using software known tothose of skill in the art.

[0239] The amino acid sequence represents a primary structure thatencodes the information necessary to form the secondary, tertiary andquaternary structure of the protein of interest. The software looks atcertain parameters encoded by the primary sequence to generate thestructural model. These parameters are referred to as “energy terms,”and primarily include electrostatic potentials, hydrophobic potentials,solvent accessible surfaces, and hydrogen bonding. Secondary energyterms include van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

[0240] The tertiary structure of the protein encoded by the secondarystructure is then formed on the basis of the energy terms of thesecondary structure. The user at this point can enter additionalvariables such as whether the protein is membrane bound or soluble, itslocation in the body, and its cellular location, e.g., cytoplasmic,surface, or nuclear. These variables along with the energy terms of thesecondary structure are used to form the model of the tertiarystructure. In modeling the tertiary structure, the computer programmatches hydrophobic faces of secondary structure with like, andhydrophilic faces of secondary structure with like.

[0241] Once the structure has been generated, potential binding regionsare identified by the computer system. Three-dimensional structures forpotential binding molecules are generated by entering amino acid ornucleotide sequences or chemical formulas of compounds, as describedabove. The three-dimensional structure of the potential binding moleculeis then compared to that of the MSC protein to identify molecules thatbind to MSC. Binding affinity between the protein and binding moleculeis determined using energy terms to determine which molecules have anenhanced probability of binding to the protein.

[0242] Computer systems are also used to screen for mutations,polymorphic variants, alleles and interspecies homologs of MSC genes.Such mutations can be associated with disease states or genetic traits.As described above, GeneChip™ and related technology can also be used toscreen for mutations, polymorphic variants, alleles and interspecieshomologs. Once the variants are identified, diagnostic assays can beused to identify patients having such mutated genes. Identification ofthe mutated MSC protein encoding genes involves receiving input of afirst nucleic acid or amino acid sequence encoding MSC proteins, e.g., asequence selected from the group consisting of SEQ ID NOS:1-9, andconservatively modified versions thereof. The sequence is entered intothe computer system as described above. The first nucleic acid or aminoacid sequence is then compared to a second nucleic acid or amino acidsequence that has substantial identity to the first sequence. The secondsequence is entered into the computer system in the manner describedabove. Once the first and second sequences are compared, nucleotide oramino acid differences between the sequences are identified. Suchsequences can represent allelic differences in MSC protein encodinggenes, and mutations associated with disease states and genetic traits.

[0243] MSC Genotyping

[0244] The present invention also provides methods to genotype ananimal, including a human, for an MSC gene or protein. Typically, suchgenotyping involves a determination of the particular sequence, allele,or isoform of an MSC gene or protein, using any standard technique asdescribed herein, including DNA sequencing, amplification-based,restriction enzyme-based, electrophoretic and hybridization based assaysto detect variations in genomic DNA or mRNA, or immunoassays andelectrophoretic assays to detect protein variations. The detection ofparticular alleles, sequence variations, isoforms, etc., is useful formany applications, including for forensic, paternity, epidemiological,or other investigations.

[0245] In addition, the detection of certain alleles or protein forms isuseful for the detection of a mutation in an MSC gene in an animal, andis thus useful for the diagnosis of mechanosensory transduction channeldefects in the animal. Such mechanosensory defects may underlie any of alarge variety of conditions in animals, including conditions associatedwith impaired hearing, touch sensitivity, proprioception, balance, andother processes. In addition, mechanosensory defects may be associatedwith a loss of contact-inhibition in cells, and thus may be associatedwith cancer in the animal.

[0246] In particular, it has been discovered that mutations thatintroduce a premature stop codon into an MSC gene within the ankyrinrepeat region, or mutations that remove or substitute a conservedcysteine residue between transmembrane segments 4 and 5 of the protein,result in a dramatic decrease in MSC activity and are thus usefulmarkers for such analyses.

[0247] Pharmaceutical Compositions and Administration

[0248] Mechanosensory transduction modulators can be administereddirectly to the mammalian subject for modulation of mechanosensation invivo. Administration is by any of the routes normally used forintroducing a modulator compound into ultimate contact with the tissueto be treated, such as the inner ear or other mechanosensory tissue. Themechanosensory modulators are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such modulators are available and well known to thoseof skill in the art.

[0249] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985))

[0250] Kits

[0251] MSC proteins and their homologs are useful tools for identifyingmechanosensory cells, for forensics and paternity determinations, forexamining mechanosensory transduction, and for diagnosing mechanosensorydefects in animals. MSC specific reagents that specifically hybridize toMSC protein-encoding nucleic acid, such as MSC specific probes andprimers, and MSC specific reagents that specifically bind to the MSCprotein, e.g., MSC specific antibodies are used to examinemechanosensory cell expression and mechanosensory transductionregulation.

[0252] Nucleic acid assays for the presence of MSC encoding DNA and RNAin a sample include numerous techniques are known to those skilled inthe art, such as Southern analysis, northern analysis, dot blots, RNaseprotection, S1 analysis, amplification techniques such as PCR and LCR,and in situ hybridization. In in situ hybridization, for example, thetarget nucleic acid is liberated from its cellular surroundings in sucha way as to be available for hybridization within the cell whilepreserving the cellular morphology for subsequent interpretation andanalysis. The following articles provide an overview of the art of insitu hybridization: Singer et al., Biotechniques 4:230-250 (1986); Haaseet al., Methods in Virology, vol. VII, pp. 189-226 (1984); and NucleicAcid Hybridization: A Practical Approach (Hames et al., eds. 1987). Inaddition, MSC protein can be detected with the various immunoassaytechniques described above. The test sample is typically compared toboth a positive control (e.g., a sample expressing recombinant MSCprotein) and a negative control.

[0253] The present invention also provides for kits for screening formodulators of MSC proteins. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: MSC protein, reaction tubes,and instructions for testing MSC activity. Preferably, the kit containsbiologically active MSC protein. A wide variety of kits and componentscan be prepared according to the present invention, depending upon theintended user of the kit and the particular needs of the user.

[0254] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to one of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

EXAMPLES Example I Chromosome Mapping and Positional Cloning of MSCGenomic Region

[0255] To identify mutations with potential roles in mechanosensorytransduction, a genetic screen was carried out to identify mutations inDrosophila melanogaster that result in uncoordination phenotypes. Thisscreen yielded mutations in numerous genes. Further characterization ofthese mutations using electrophysiological methods determined thatseveral of the genes also reduced or eliminated bristle mechanoreceptorpotentials (Keman et al., Neuron 12:1195-1206 (1994)). One of thesemutations, responsible for the nompC (for no-mechanoreceptor potential),present on the second chromosome, abolished nearly all of themechanoelectrical transduction in mutant cells. Flies with this mutationare uncoordinated to the point of lethality. Based on these phenotypes,the gene underlying the nompC mutant was identified as potentiallyencoding a protein playing a central role in mechanosensorytransduction, such as a mechanosensory transduction channel.

[0256] To determine the position of the nompC gene on the secondchromosome, nompC mutations were genetically combined with varioussecond chromosomal deletions, and the resulting transheterozygous flieswere screened for lethality. In this way, the chromosomal position ofthe nompC mutation was mapped to a small interval on the left arm of thesecond chromosome, corresponding to map positions 25D6-7.

[0257] To physically isolate DNA in the 25D6-7 region, the proximal-mostclone from a chromosomal walk in the nearby 25D 1-4 region (George &Terracol, Genetics 146:1345-1363 (1997)) was used to probe a Drosophilacosmid library (Tarnkun et al., 1992). Overlapping clones were used to“walk” to the area that contained the nompC (MSC) protein encoding gene,by mapping the cosmid clones to genetic breakpoints. At the same time,the cosmids were tested for the ability to rescue the nompC mutantphenotype. One cosmid was found to rescue the lethality, uncoordinatedbehavior, and physiological defect of the nompC mutation. This cosmidwas thus determined to likely contain the MSC protein-encoding gene.

Example II Sequencing of the Rescuing Cosmid and MSC Gene

[0258] To determine the sequence of the cosmid containing the MSCprotein encoding gene, the genomic DNA insert from the cosmid wasisolated, sonicated, polished, size-selected, and the resulting 0.7-2 kbfragments subcloned into plasmid vectors. Plasmids were purified andanalyzed for the presence and size of inserts, and 123 clones withinserts of greater than 0.7 kb were sequenced. The sequences determinedfrom these inserts were used to assemble large contiguous fragments,which were extended by designing ad hoc primers from the ends of thefragments and using the primers to read additional sequence from thecosmid DNA. In this way, the entire 33.6 kb cosmid insert was sequenced.

[0259] The MSC protein-encoding gene was identified and characterizedwithin this 33.6 kb cosmid sequence using exon analysis, BLAST searches,and secondary-structure prediction programs. These analyses establishedthat the MSC gene is a large gene comprised of 19 exons, encoding aprotein containing at least 21 ankyrin repeats and a set of as many as11 transmembrane domains (6 of which show significant robustness), thatis weakly related to the TRP family of epithelial cation channels (see,for example, Montell, Curr Opin Neurobiol 8:389-97 (1988)).

Example III Sequencing of nompC Mutants

[0260] To assess the molecular defects of the nompC mutants, we used PCRto amplify the genomic DNA encompassing the nompC locus from flies withone of four mutant nompC alleles. In this way, all four alleles of thenompC gene were amplified in approximately 2 kb fragments that coveredthe gene interval. These fragments were then sequenced. All four of thenompC alleles showed mutations in the coding region when compared to thesequence of the cosmid and to the parental, wild type DNA.

[0261] In three of these alleles, the nompC (MSC) polypeptide encoded bythe mutant gene was prematurely truncated in the ankyrin repeats by theintroduction of stop codons. The fourth allele had a missense mutationbetween transmembrane segments four and five, resulting in a C to Ysubstitution.

Example IV Identifying MSC-Related Genes in Other Organisms

[0262] To identify potential MSC-related genes in other organisms, weperformed sequence comparisons between Drosophila MSC sequences andnucleotide and/or amino acid sequences present in various publicdatabases. In this way, a previously unknown C. elegans genomic sequencewas identified as an MSC homolog. This genomic fragment was found in the“unfinished/orphan” domain of the C. elegans genome project database.Using a variety of sequence analysis programs, putative coding exons,intron sequences, candidate transmembrane domains, and homology regionswith Drosophila MSC were identified. FIG. 1 shows an alignment betweenthe Drosophila melanogaster and C. elegans MSC homologs.

[0263] Three signature sequences for MSC, based on alignment analysisbetween the Drosophila and C. elegans sequences, were identified and areshown as SEQ ID NOs:7, 8, and 9.

1 17 1 24358 DNA Drosophila melanogaster genomic nompC(no-mechanoreceptor potential C) nucleotide sequence 1 gtgaccatgttgcgggggac atgtttagta attgcaaaat cgatcaggtc tgggattttt 60 cttgggtctgctggccagta tgtaggctta cccggggata attcgctctc ttaatgtgat 120 aatattaatctcagaataat gaaaatgtca ttggtgtggg aaaatgtggg aaattgtcaa 180 ggaacgtagagagtaacatg gtaattctat attttatttt tatttttctg atggtaaaaa 240 agttctagctttatagtaat aatatcatta ccttgagtta gtaagattta aaaaataaaa 300 taagctgcattttaaaagcc acctttactg gttagacgac agcaacgata agataagttt 360 acatttttgctacttgcatc acttgttgcg gcatcactga taagcaaaca gacataattc 420 gcgtggctggaggttttcct gattcctatc gctatatttc tgctcttatc atgcccccaa 480 aaaagttctgcccatactca aagaattgct ttttatttag ttgaccttgt tgtcaaatca 540 gcaaggcatatttatatctg caattggaac tacaattgat gcataagaaa tgaggtgttt 600 gtgaatatctttgaaactga aacgaaagtt agtaacttag tttagtaact agtttgttta 660 gatataagtgagttataagt tgaattaaaa gaaggatcac ttcttctagt attgataaaa 720 ccatttattatacagagagt tatagaagtg gctccatgta acctagacta gccaaaaaac 780 tattaggcattcattttcct ggccacttgg gattttcgcg accagtcagc aaggatgaca 840 tactcccaattgcgtctgtt gcccatttgg gtttcccacc ggcacttaac gacgttggaa 900 atcccaacgaaacttaagag tagcgtccag attttggcgc caaaaaaggc ggtattattc 960 ggattcaacaattgtaaaca aacgcttgcg cggatgccac ttggctctta cctctgattt 1020 ttcgcaggagcgtcttgggt ccttcgagtt tggagcttcg tcgtgttgcc agagctacca 1080 aaccgagtggagggccgagt ttttccgctc gagcgccttg ggaatagtcg actctgtgaa 1140 aatgggactggcaaatcaga aactcgcaga cgctcgtggc aaacggttga tttttttctc 1200 gtcgctccgaaaaaaggcaa aatagtaggc aacctgaaat ccagagttgt agttggggac 1260 tcttttggccaaaatacaag gaggagaaaa atagaaaata ataaaggggg caccgccgtt 1320 aacgcacacgcaaccgaagc cataaagggg ctaaacatat aaatttgtgt agtaaaagtg 1380 aagaaagcgaaagaatcaaa gtggaataat agcgagtgtt tttcggtttg ctagtgtgtt 1440 tctgagtcggagtttgtgtg tgtgtgtttg tgtgattcct agtgtgtctg ttgctgttgc 1500 caatgaaaatgcaaattgtt ggtaacaaat attggtaaaa tgcggaggcc gtaggaattt 1560 gtgcaatgcgagtgcgaagt gaaggagccc gaaactatgc agctaaaaac ccgccatcct 1620 accccgcatcgaatcaataa taatacaata acccaaacgt attacacgga taatggcagc 1680 ataaaccagttaacatccga cagtgtttcc gcctaaccat cgagcaccta gctcatcccc 1740 cctgccaccaacccttcgaa aaatccccat gatcagcgcc ggattgtgga gcagtaacta 1800 gcgaggcataccaggatgtc gcagccgcgc ggagggcgtg gcggtgggcg tggcggcgga 1860 gtgggtcgcaaaaccccctc ctcgctgacc ggcccaccgg atgagtcggc tacgcccagc 1920 gaacgggctacgcccgccag caaagcagac tccgatccca aggacgatag ctcgagcaat 1980 ggcgacaagaaggatatgga tctttttcca gccccaaagc cgccgagtgc cggcgcctcc 2040 attcgggacacggcgaacaa ggtgctcgga ttggccatga aaagcgagtg gacgcccatc 2100 gaggcggagctcaagaagct ggaaaagtat gtggccaatg tgggcgagga tggcaatcac 2160 ataccgctggccggcgttca cgacatggtg agtactgtac agtgaagtgc cgcgaggcgg 2220 gctttccggctcatttgcct cgttttgtaa aatcaattgc gagccaaagc gggaatagga 2280 agcgaaataaatacaggaac aggtccaaca ctcagcgaaa aatatggtaa attaaatgta 2340 tacctagagaaggattatca atagttttaa taaggttatt gaaatcttta aaactataat 2400 ttctatggatcttttagttg tatttatttg aaaaatttcc ttaagttttt gtgtaatatt 2460 tccctgagtgtatgcgatgt agaaacgtcg cccttatcaa cgtcggcggc attttcccat 2520 ttctggttgtttaccagcca aaataacgac acaggaactg gaggccagaa aacagagcac 2580 accatggtttggccaaaaaa cagaggctag caaggaaaag cgcccaaaaa aaaaaaaaac 2640 agagaacagcgaatgttatt tgatagctcg gcccaaatgt tttggctgcc aaggcgatgg 2700 ctttggtggcattcggtttt gtagctccaa gttcctgaag cgtcctgcca caagttgcgc 2760 cgtatacgctttggggttag ccccccgtcc gaccgataaa ctcataaaac atcgaagaat 2820 tgaagcgcttcgatttcaat ttaccataaa cgctatgaaa cggagaagtc gttgacataa 2880 aattaacgttgcaccgctaa tgaaatgcgg ggaggtgtgc ggcgaaaggg ttgaaacttc 2940 ctggcagggtttttctttta cttttttcct ttcctttttt tttttgtgtg gtactatata 3000 tcccaactagatgtgcaggt tgtctgctag actagactta cgacgagacg gtatttgcat 3060 aaatatagcttggagttgag ctatttttgc cttgattatt tccgctttcc cagaacgggg 3120 gtctttattcggttcttgac ttgatgggct tgctcttgat ttcgttttaa ttacgagcca 3180 acgagcttataatatcacat ccagcttatt agccgaagga ttctaatgca ataaagatga 3240 atttaaatggccaagttgct tttcaatgag gtcagcgggt tggaaggaga gtaccatgta 3300 ttggtactatgttattgtgt ttaaaatgtg catatattaa tattgtatta ttcttacctt 3360 aagcttaagtaatccccata catttccatt gcagaatacc ggcatgacgc cgctgatgta 3420 cgcaacgaaggacaataaga cggccataat ggatcgcatg attgagctgg gcgccgatgt 3480 gggagcccgcaataatgtga gtcttgagcg ggaatagggc aggaataatt taaagcacct 3540 tagccaactccccacggtgt tggtgccaaa tatagaagcg gcccagctgt ttaagccaac 3600 ggcggcagcaaaagccgcta aaaatgtgtc aaatcaataa aaaccgcata attaaatctt 3660 gagcgggggcgttggtgggt aaactcgtgc acccacttct acgcacgatt ctcacacgcc 3720 gcccaccacggtcaatactt caattcggca atacctccct gccgcaatgg gtcaacttgg 3780 caggacttggccaatgggta gttcgcttca tttgactcca gttgagtcaa gttttccagc 3840 acgaatgggaatttcctcaa gaaaaagaaa tactaacaca ttgcttttat tttcatttta 3900 taactgctaacaaaaaatta taaactctta tttatagaaa actaaattat tattgggcac 3960 ccctcgtttttaagtggctt aaagttcgaa cttaactttg gtttttaaag aaacagcaag 4020 tattactcataataatgtaa ctcaacaaaa gagttttccc aaagagtaga gatgtaaggt 4080 catcgctgatgactatcctg atttccccag taatttacca tcgtgattat ggccaattct 4140 tttttttttttgatgtcagc aagtgaagtg agccaggttg gcatcgccca ttaggccaag 4200 ttgctaacaattggtcgaat tcgccgacca gcttgctttg catgccgcaa ttacttagca 4260 catttcatttgaagtcgctt tcttggctgc ccattcacat gtccttacgt atacgcaacg 4320 tactttatttcggtgctagc ggcgataaaa atccttgacc taattacaaa ataattgttg 4380 ccaaaccagtgcagacatgg cgaattgaat taccaaaaca aacacagaaa gttcaatttt 4440 cccttcctccttgaaaatgt ttctcctaaa agattaaaga gtgtgtaggg aaaatgttaa 4500 aggtaaatttgcacatgaaa gtcataaaac attaactagc cgggagttac aagctaagca 4560 tgaaaataaaacactcgata agactttata tgagtataag aatttatttt cgttttaaca 4620 ggacattcattacacaaatt ttgccaatga tacttggtgt tttaaaatat tgagaaaatg 4680 ttgtccaaactgcaactaaa aaccacatat atattaatta attatattta atataaactt 4740 tccctttttgcaacacaatt aattatgata attattcatt ttaaaactgt tccatttgga 4800 tgattgttccctcttgttgt tcagctaatt aaatattatg atatcatttt cgtgagttta 4860 tacaaagcgcacctttttga aaaccattac ctcatctgta taattactct tttgttttta 4920 taaaacaaatgtcacttcgt gaccaaatcg gataatttcc cttacactga ccaaatgaat 4980 taaaaactgagaaatgttta ttgcatttac aattcgcaac ttatctaact gtcaggtctg 5040 gtccaaagtaatacccaaac aacacgacag gaccaggacc tttatggcca ttataaagga 5100 tactcgtatgatgtaacgcc gtggtaatta acatttttaa cttttcaact gcaaggtggc 5160 agactgcttttttttcggca ctcgacttgg aggcgtgctc gcaacacctc tttgcaacgt 5220 aaaagccaattaatcaagca catgactccg atgtacgccc agttggccaa aaactccatt 5280 tgacctttcgagtgtggccc aaaccggaga cctcgacgtc ggccccgact tccgctacat 5340 ttttatggccagcggcgtca ttaatatgca attttaatta aattcaagtg gaattcttca 5400 cgcagtgacccctgcatatg tgtgtggcga tgacagcgtg aactaaaatg aggaataaaa 5460 acgccaattcatttgtcaag ttgcctcagt gcgtgagtga agtaatctgc cccatccacg 5520 caaaaaaaaagcaaattaat tcacttcatt agaaagtggt gcacatgcaa gaaggtggag 5580 ggattaagccaaatgagcac cgtaatgagg acttgcaatt attccaaaga aggtgtgtga 5640 catcgccagaaaatgacttc atggcttcca cgcgactatc cccgagtatc tctgggccgt 5700 aaaaacaaaacacccacgaa actgggtcga cttcgtacac ccttatccac ccaaccttat 5760 cccttttccatttggcaggg caaaaatgtg ctggaaaatt tgcgcttccg ctttggtttt 5820 gtttccggtttttcctttcg accagccaag caaacgcaaa cacaagcgca caaacacaca 5880 agactcgaaaacgaactcga acctggctca aaagtatgca aaacagcgcg tgaaatatta 5940 tctgtctaccttggacgcca atgcaacccc aaaccagcag cgattccgcc caccgcgcca 6000 agtggctgaaagtttacttt gctttttctt tagggccaac acgtcttgga tgggctttct 6060 ggacatgtgtcaaagccgtc gactccgagc gccaacttgc gttgtatgca aattagcagc 6120 agctgcggccagaaatagtc gcaaataaac cgcagggaac tcgaatttca cacggcacga 6180 agcccacacacactgactta agtgggaaag tttgaaatac ccatttggat tctaggaatt 6240 gtaaaaaatcatgtgcaaga acacatagaa tgtataaata tagaattatt ttaaatggca 6300 taacttctggtattctccta attttttaac atataatcta aactaagtat tattttcctt 6360 tcactatttttattaactag aaattcgtat ccttttatgt tgaattttgt agactctgtc 6420 tgcacttaccaacctgatga cagggccaaa agcacccata catatatgct aaaccagttc 6480 acttccgttttcggggctaa gaactgtggg gaggcttagt tataaattag agccatggtc 6540 cgaggtccgagcatacgggg cgtatgtgta acacgttgcg ttatggctta ttatataagg 6600 caataaatatggccaaatgc ccccgattca tatgtgactc acttggctat tagctggcgt 6660 taaactaagcactccatgtc agacgttatc ttaaagcact tttcgttacg tttcggtgat 6720 ttgctcagggtcatattttc ctagccgcat tgttttatat ttcttttcgg gttttcctgg 6780 tcgccattgatgcagttttt gcatgtgagt ttgcggctgg gctgtggcca ttaagaaaac 6840 cccgtccgtaagtgaaagtc cgcatgcaag attgtggctt aagtaatcaa ccactccctt 6900 ttgccccgttagccgcatgc aaaaccgact gactttgacc cattgaactg acccagctct 6960 tttggtgtgggggcgtcagt ttcctgccaa tgaattgcaa ttgatttcct ccgttcttct 7020 cttctcttctctttcaggat aattataatg tgctacatat tgccgcaatg tattcgcgtg 7080 aggatgtcgtcaaattgttg ctaacaaaac gcggcgtgga tcccttctcc accggtggcg 7140 tgagtattccaatagcttta tatactacat atatacgtat gcgccccaag aaagtgttac 7200 cccaatagttgaggtagcga cacgtcaggc gacacactca atactcgagt tcctactttc 7260 gagtcaatgaaatagctgca taccttgggg ctgctgtcag cccgattcgc aggcaatttg 7320 cggctattagacgcatactt cacctggctt cgaaagagaa gaaaaaaaaa aaacctatcc 7380 aaaggtcagagccatgcgaa gatgcaactt tgaggctcgc atgttgcatg ttactttggc 7440 gggaccagcaattaactggc gacaaggtta agatggtaat gtctagggcc cgcttaagaa 7500 cactttaagacctgaaaaca aatttaaagt aaccctaggt ttcacgaaaa actttactca 7560 tcagattaaacagaaattta agcttagata ccgtcattaa aattaaaatt taacattttg 7620 catgatttccaagtctgact tctgtttaaa tactacaatg tataaatatt aaagtctgag 7680 caagattagtgacaccatct ttatattgtc taaaatcata aagcgttaac catttaatac 7740 aatgcattttctcataggta acatttttaa caaaatatat gatgatcaca tcgtcaagca 7800 ttttggcaattatttctcca agtttatttc tcgtgtcggc attaatttgc ttttctttat 7860 ttttttctcggccgcattgg gttttcgaga cttggttatt tagggggcgt gcgccttgcc 7920 caaattactgatggttatca gaagagagct ctaagcacgt gtgggagcga gagaagtgga 7980 gctgcggaagcgagacagac agatgcaaac ttttgtttta gcaacagcca agtttgaagt 8040 gttccgttagcgtgtgtgcg tggcaaaaag gactcccaca tccacaaccg acacctgccc 8100 cccatgttgcctacacctgc tgctcgacca cccctccccc accatcacct atatacacct 8160 ctctcgctcactcccgcagc ggttgtcggt gggagttctt tattatgctt ttttcgggct 8220 gtcaatctgtgatatgagcg ggagaggcca aaaaagaaaa atgacacgaa atgtgcttat 8280 aaacgcaaaaacgagccact tgcctattca gtagcaaatg gaattttgaa gcgaataggg 8340 aaacagtttgccagtttttt aggtgccaac attaaccaca cagtagtgca catagctgca 8400 tattaattttggctagaaaa aaagtgtaac cccagcaata agtgcgtttg cagtgtgtgc 8460 atagtttaatcgaagactta attggatttt tttccctttt cagtcgcgtt cgcaaactgc 8520 ggtgcatttggtgtccagtc gacaaaccgg aactgcaact aatatcctgc gcgctctgct 8580 cgcggcagctggcaaggata ttcgcttgaa agcggacggc gtaagtgtta ccatgtgtgc 8640 ttgtgattgagtgtgccagt gtggctgtgt gtgtgcgacg gagagccaca agtgttggcc 8700 gcccaattgatgccgcttta tctccactag tttatgatag ctaagccacc caaatgcaag 8760 ccgatgtgaagtcaagtact ctcgacagcg gtgccaggcg gtgccgacgt aaacaaagac 8820 ttaataaaaatcaccaaaaa atatatacat tacaataatg gcaccaacaa aatcgagagg 8880 agttagtaacataaagcaaa caaaattgtg tggaaaaatc gatatgcaaa actgctcgcg 8940 gtaaatgcatttcgactggc tgtaaatcag aaaaggccca aaaaagttaa tgcggctatt 9000 acacagcgaggaattgaata ggtaattttt gagtcaattt tagcttataa tttgtggtac 9060 ttttatgaatttttttaaaa tttttatttc aaattattag agagctaata tatttgaatt 9120 atgcttatataacttaaaat actcaaaatt tatagacagc aataaagtat gggatctgca 9180 acacatctttttctacactg tatcaataag tagctctcac cacagtgggt aggctccagc 9240 gagctttgaattaccatcga agcagttgtc tccgcctgat gaacttgctg gggctaaccg 9300 agctccagatccctttttcg agctcccccc ttggaaatct gaacagaaat gcggaactat 9360 ttgtcgcatcacgtgccccg ggtgaaaatg cacaggcgat atttccatta cgcacgcgaa 9420 gaaagcgcataaatttccaa cgaattgcta tcaagcgatt gtaaggattt ggggtatatg 9480 ggggctgattgagggaatcc cgggtgccac cgattgattg tctagacaaa atgggtaacc 9540 cacctcgatttgtgcctcga gggctgcggc aaatggcaaa cagcaacttg atttaaatca 9600 attagagagaggtggaatgg cactgtcagg cgaaattagt cggatgaagt atttagcttt 9660 cgatggcattcagttcgatt cgtttcgatt cgcttttctt ttttttttct acacgcattt 9720 ccggtgtgcatatacatgca aatatatata ttgtatgtgt gtggatagta ctgtagtttt 9780 cccccgcgagggcgctcaac tcgttgccaa caacaaacaa atataacaaa gcgaggaaaa 9840 ctctaccgaaaaaagggggt caagtcgctg tacaacttga tttactcgcc tttcctggca 9900 gatagggataatggctcccc gtcacgcccc cctcttacga ctcgccccca aaaggtagtt 9960 ggttgcaagttggagcgcca aagttgcgaa cttggctaaa aatagcgaaa catgttgccg 10020 ttaacacttgaggctcgaat tggctaattg gatatttatg attatatgtt cgcgagtgtg 10080 aatggatgtgtgttcgctgt ccttatctta attatatttt atactatata taacctatct 10140 ctaacctagcgtggcaaaat accattgctc ctggccgtgg agtcgggcaa ccagtccatg 10200 tgcagggagctcctggctgc acaaacagca gagcagctca aggtaagtaa tctgtgaact 10260 agcagataagtttacccact tattttaaaa cctaaaagtc tagttgcagc ttatattgat 10320 ttaaatagaaacactgaata catcatctag ttaataacca aaaatgtcaa cagtatgagc 10380 cattaaaagcataaaatgct aatttcttat accatctacg catctaactg atttcctaac 10440 taggaccaagaaattgttga ttttataatc gccacgatag tgtcaatcaa actgtccatc 10500 tgagctgtcggaaaatgtcc acaaggttct taaagccttg aactgtccaa taaccaagcg 10560 tgtaaataaatcaaaaatgc aaatttaccc tgctcacctg tgcgtacagg tgcattgcaa 10620 gtgcaacagtgcgcgacatt ggcaaagttt gtgcaatttt caatcagaag ttgaagtgca 10680 acacaccaagagcagtgcgt gttgattaaa ttaaccaaag ggctacggct cgcttcaggc 10740 caagggttcaagcccaagtt aaagttaaag ttgcgcctga ctttggccgc tggctgagca 10800 cgcaatcagccggcaaaaca gccgtaaact gggtcaaaac tgaggcgaaa acgcagctaa 10860 gatgggaagggaatctgatt tgcatagccc aaaataaaat gtcgaaagtg aaatgcagca 10920 acactaaggaaaaatttaag taaattattt aaaaatattt aaacaatgaa gctatgaagc 10980 tctagcaaagataccaattt agttagggaa tatcattata atttgtcaca tagttaatta 11040 atttcaagcataggagcaat tatgactttg caattatata aaaacatttt tgtgaagtgc 11100 accctttcatgttaaatttt ggatttattt tttcgcaggc aacgacggcc aatggagaca 11160 cggccttgcatttggccgcc agacggcggg acgtggacat ggtccgcatc ctggttgatt 11220 acggaacgaatgtggacacg cagaatgggg agggccagac gccacttcat atcgcggccg 11280 ccgaaggcgatgaggctcta ctcaagtact tctatggcgt gcgcgcctca gcgtccattg 11340 cggacaatcaaggtgagtct gtgggaatgt ggagcaagga aaagcatgtt gcaaatcgtg 11400 tttgaccttgatataacaca ataaaaatca tgaaattttc acttctcaat agaagctagt 11460 gattataaagtggaggtata aagtatatgt ttgtggcgcc cccggttgga ccgagctcca 11520 gacatacgaatgtccgtctt gatgattaaa atttatatat atatatatgt aataccctat 11580 agatcgcactccgatgcact tggccgccga gaatgggcac gcgcacgtca tcgagatact 11640 ggccgacaagttcaaggcga gcatcttcga gcgcaccaag gatggcagca cgctgatgca 11700 cattgcgtcactcaacggtc atgctgagtg cgccacgatg ctcttcaaga agggcgtcta 11760 cctccatatgcccaacaagg atggagcccg gagtattcac accgccgccg cctatggtca 11820 cacgggaatcatcaacaccc tgctacagaa gggcgagaaa gtggatgtga ccaccaatgt 11880 aggtgggataatgtattaag ggataatcgt attaattcca cactctttgc aggataacta 11940 tacagcactgcacatagccg tggaatcggc taagcccgcc gttgtggaaa ccctgctggg 12000 atttggagcagatgtccatg tccgtggcgg aaaactacgt gagaccccgc tgcacattgc 12060 ggcacgagtgaaggatggag ataggtgtgc cctcatgttg ctgaagtcgg gagccagtcc 12120 aaatttgaccacggatgact gtctgacccc cgtgcatgtg gcggctcgtc atggcaatct 12180 ggccacgttgatgcaactcc tcgaggacga aggagatccg ctgtacaaat cgaatgtgag 12240 tagattattagaatagaatg ataaacgctt gaattaaaac ttccatttta tagactggag 12300 agacaccgctgcacatggcc tgtcgtgctt gccacccgga tattgtgcgt catctcatcg 12360 agacggtgaaggagaaacac ggtccggata aggccaccac ctatataaac tcggtaaacg 12420 aggacggcgccacggcgttg cattacacct gccaaatcac caaggaggag gttaagattc 12480 ccgaatccgacaagcagatc gttcggatgc tcctcgaaaa tggtgcggat gtcacgttgc 12540 aaacgaaaactgccttggag accgctttcc actactgcgc cgtggccggc aacaatgatg 12600 tgctgatggagatgatctca catatgaatc ccacagacat ccaaaaggcc atgaaccggc 12660 aatcatcggtgggctggact ccactgctga ttgcttgcca tcgagggcac atggagctgg 12720 tcaataatctactggcgaat cacgctcgag tggatgtctt cgatacggaa ggacgatctg 12780 ccttgcatttggctgctgag cgaggatacc tgcatgtgtg tgatgccctg ctgaccaata 12840 aggcttttattaactccaag tcccgcgtgg gacgcactgc actacatctg gcagccatga 12900 atggatttacgcatctggtg aaattcctga tcaaggatca caatgcagtt atcgatattc 12960 taacgttgagaaagcaaacg ccgctccatt tggcggcagc cagcgggcag atggaagtct 13020 gtcagctgctcctcgagctg ggcgccaata tcgatgcgac ggacgatctg ggccagaagc 13080 caatccacgtcgccgcccag aacaactact ctgaagtggc caaactcttc ctgcagcagc 13140 atccatccctggtgaatgcc accagcaagg atggaaacac atgtgcccac attgccgcca 13200 tgcagggatccgtcaaggtg atcgaggagc tgatgaagtt cgatcgatcg ggtgtgattt 13260 cggcgcggaataaacttacg gatgccacgc cccttcagct ggccgccgag ggcggacatg 13320 cggatgtggtgaaggctctt gtgagagctg gtgcctcctg caccgaagag aacaaggcgg 13380 gattcaccgccgttcatctg gcggcacaga atggacatgg tcaggtcttg gatgtgctga 13440 aaagcacaaactcactaagg atcaatagca aaaagttggg tctgacgccg cttcatgtgg 13500 ctgcctattacggacaggcg gataccgtgc gggaattgct gaccagtgtt cccgccaccg 13560 tcaagtcggaaactccaacg ggacaaagtt tatttgggga tctgggcacg gagtccggaa 13620 tgacaccactacacttggcg gccttttccg gcaacgagaa cgtggtgcga ctgctcctca 13680 actctgcgggtgttcaagtg gatgcggcga ccatcgagaa cgtaagatta cctgcatatc 13740 tcttctgttcagaaaccatt aacacaacaa ttgattctac agggctataa tccactccat 13800 ttggcttgcttcggtggtca catgtcagtg gtcggtttgc tcctaagtcg gtcggcggaa 13860 ctcctccaatcgcaggatcg taacggcagg acgggcctgc atatcgccgc catgcatggc 13920 cacatccagatggtggagat tctgctcggc cagggcgcgg agatcaacgc aaccgatcgg 13980 aacggttggacgccactgca ttgtgctgcc aaagctggcc acttggaggt ggtgaagttg 14040 ctgtgcgaggcgggtgcctc gccaaaatcg gagaccaact acggttgcgc cgccatttgg 14100 ttcgccgcctccgagggaca caacgaggtc ctgcggtatc tgatgaacaa ggagcacgac 14160 acctacggcctgatggagga caagcgattc gtgtacaacc tgatggtggt gtccaagaac 14220 cacaacaacaagcccattca ggagtttgtc ctggtatcac cagcacccgt ggatacagcc 14280 gccaaactgtccaacatcta catagtactc tcgacaaagg tgatttagct aaaggatctc 14340 tatgcacttaactaaactaa ctaactaaaa cattttgatc tctttaggaa aaagagcgcg 14400 ccaaggatctggtagcagct ggcaaacagt gcgaggcaat ggccacggag ctcttggccc 14460 tggcagctgggtcagattcc gccggaaaga tccttcaagc caccgataag cgaaacgtgg 14520 agtttctcgacgttctcatt gaaaatgagc agaaggaagt gattgcccac acggtagttc 14580 agcgatacttgcaagtgtgt gatattattg actagcttag atcttaactt attgagattc 14640 tgatatgtatccttcttcct acttttagga actctggcat ggctccctga cgtgggcatc 14700 ctggaaaatccttctgctgc tcgtggcctt catagtctgc ccaccagtgt ggattggatt 14760 cacattcccgatgggtcaca agttcaacaa ggtgcccatc atcaagttca tgtcgtacct 14820 aacctctcacatttacctca tgatccacct gagcatcgtg ggcataacgc ccatttaccc 14880 agtgctccgattgagtttgg tgccctactg gtacgaggtg ggtcttctca tctggctgag 14940 tggattgctccttttcgagc tgacgaatcc gtcagataaa tcgggactgg gatcgataaa 15000 ggtgctcgtgctgctgctcg gcatggccgg agtgggtgtc catgtctcag catttctatt 15060 cgtctccaaggagtactggc caactttggt gtattgtcga aatcagtgct tcgcgttggc 15120 cttcctgctggcctgtgtgc agatcctcga ctttttgtcc ttccaccacc tattcggtcc 15180 ctgggccatcatcattgggg atctgctgaa ggatctggct cggtttttgg ccgtcctggc 15240 catctttgtgtttggctttt ccatgcacat tgtggccctg aatcagagct ttgccaattt 15300 ctcaccggaggatctgcgca gcttcgagaa gaagaaccga aatagaggct acttcagtga 15360 cggtaagtcgaaacgtttgc tttgctttct ccagtctact tttcgaattt ttgtttcgaa 15420 ctttttgttttcatttggaa tgtttttgca aacttcctct tttgaacgtt caatgtgtct 15480 tgataagtatctgtgtctgc cttgaatgaa aagcccctct aatcaatgtg cgctcgatgt 15540 ttcacataagtaaaataaag caaaaaagaa ccaacttcaa ccacataata caacaattgc 15600 atgctcaacaagtacaaaca acccgaacct ccaaccttga tgtcgtaatc cccgtccacc 15660 cctccaccaaaagacctcca ctaataatgt tctccctctg atcttaaccc ccaactgaat 15720 atcttaactgaattatccga atggaacaga tgacatgccc acaccccgac ctccgccggt 15780 ggagaattatgtcgatagtc gcttcagcga attccgacga aagcacaagg acgaccgtaa 15840 gtctcctaccatccacaact accaaccctt actacccccg catttgcatg gccccccttt 15900 ccgggggctgccccgccccc ttaacccaac aatgccggaa tccaaaccgt tgcgttgccg 15960 ccttcgatgttgtgcgtaaa gtgttaatgt cgtttgtttt ctagttccct ggaggaacat 16020 ccacaagtccgcactcgctg ctcgaaatcc cctcgccttg ctagtttcag ttactttcgt 16080 tttgaggcatgttcgcggga aaatcccttt tccgcatcct cgatgttgtg gatctgtgtt 16140 tatataggtatccatgcgcc aagctttatt acttagtttg gagtatcgtt ttataccttt 16200 gcttggatcaattttaattt atatgtattt ctttatgtat ttttaagtga catataaata 16260 caaataaattattaagaatc agaatttaaa accataattt attctcatta aattcaatca 16320 ttattatttcaaaaaatcct agatctgtgt ccgatattat tttctttact atatttgtta 16380 ttctttttttaagttagatt ttttatcgat gtgtaaccag agcgatatcc attagaactc 16440 tgtacaaactaaaaattcca gtaatgcatg ttgatgtttt tatccagtca atccaaacca 16500 aaatcaaacaatcaatcagc aatatcgata taaccaatgc ccgcctgcct ggggctttca 16560 gcttgcgccgcttgcccacc accaaattct gcacaatcga aacaatcgag accgatcgaa 16620 tcgaatcgataacgaaaaac gataacgcta ctgataccga ttaccgatgc tcgtattcgt 16680 gagtcattcgaaccgctcag ctgcgaactg cgagatgctg cttttgacgt gtttaaccac 16740 tcacccgcactctccaaaat ccaaataaac ccacccataa atatactcgt ttatgtaaac 16800 ttcaaaataaccaacaaata ccaagtatta aactcgcaca cacgcctgtg ccaagccgac 16860 aatatatatacgtatatata cgctagctgc agcaatcgca atgcaatagt tcagttatct 16920 gattgtgagtaacgttccgt tcggacccat gttaggaccc atgacgccct ttctggcttt 16980 cgagcgcctcttcttcgcgg tcttcggaca gacgaccacc ctggacatca atcccatgcg 17040 acacttgcgtcccgagtgga ccgaggtgct cttcaaattt gtctttggca tctacttgtt 17100 ggtgtctgtggttgtactca ttaacctgct aattgccatg atgtcggata cttatcagcg 17160 cattcaggtttgtattgcca aggccactaa tcagtatttt ctctctgctt tccctcttcc 17220 cccgtttatttgtttcaatt ttcatttacc ggaatgctat ttgtttgtgc tttgattgta 17280 acaaccccaaaactgaccgc tccaaattga aacacaattg ggcatgaacc gaaactgggg 17340 gttggtcgatcggacaaatc aacgaaacaa aaaaaaaaaa aaaaaccaca taatcgaatc 17400 aaccaacccaacctgggcgt ccgttatctt tttatttttc aaaataattt ccacgccggc 17460 caatatatgcgtgctgtccg ggggtgtcta tttgtatctg tatctgtatc tggaaatgta 17520 tctatgggtctccgacacag tgcgcatgca tccgattaac tcgttcgagt tgttgttctt 17580 cgccgtgttcggacaaacga cgaccgagca aacgcaagtt gacaaaatca aaaatgtagc 17640 cacgcccactcaaccgtatt gggttgagta cctgttcaaa attgtctttg gcatttacat 17700 gttggtgtcggtggttgtgc tcattaacct gctgattgct atgatgtcag acacctatca 17760 acgcattcaggtagtattgc taaatgcgct tttatctaac tcgactctat ttattaactc 17820 gtactttaaccataagtata taaatttcat attgcattgt gtattaatca ttctctattt 17880 cagcataagaagtaaattta catatgaaga tgatttatat ttcttagata tataatagcg 17940 gtagttaggaagtgagctgt tttgggaaca tattgagaaa atagttaatt aatctggaga 18000 acttggcatgctctgtaaat ccatcaactg cccagacttg catcttccag gttttttcag 18060 gaaaataatgttagcaatct gagggataca attttgtgaa agtgtatctc aaagatggaa 18120 gcctgccgccttctagtgta gtacagtgca gagtagcttt agtggattag ccgccttgaa 18180 gtgtgccctgcttttgtgac cagtgttgag cgaggccaaa ccagaaagtg ttggttaacg 18240 catgcttacaaaaccttata tatagaaatc gttgctgcat gcttatatgt ctgtgtttgt 18300 cattgtctaggacttaagtc tgaagagata caccaatatg gtggttaggt tttgtatggt 18360 aattttgtgattgccatcca aaacaggcct ctgaatttgt gtatttctat tattaacaac 18420 ctgatttttgcagctcttaa gttacgtatt aacaaagtaa aaacctgtaa aatccgaggc 18480 ttctgttcacgaaactcatc ccgtttattc ctttgttctt gttctctcct atatcatgtc 18540 tcatccatccaacatcgcgc acctcgctaa ccaataataa actgaacaaa aaaaaaaacc 18600 tatgaaatactaggcccaat ccgacatcga gtggaaattt ggcttgtcca agcttatacg 18660 caatatgcatcgcaccacaa cagcgccatc gccgcttaat ttagttacca cctggtttat 18720 gtggatcgtcgagaaggtca aggtaaaatc tcaggtgacg aaggtcgcct tccagccgct 18780 gtcgctgtgtctctctctct ctatccgtat cctgtatcct gtatcttata cctgtttcca 18840 tatctgttgactatataaag tgcaactacc agaaccgatc ctgaacgggt gtagtttgct 18900 gaccttttccccaacccatt taaagcaatt tggcaacaac cgcaatgagt ttgaacacag 18960 tgaatgctttaagtgtgttg cccacataag aaaatcacct tgtcaccttg cactttctct 19020 gtaacttcaaaataggagat cgaaatatag gtatgtaaat gtttcgatcc cctacactgt 19080 atggcactttatgtccagca cttggcaccc gattgctttc gatgtaatga acatttgctg 19140 actgcgtttatgttgtgtct cttgtcttgt atgtgatcta tgtcccgtgt ctaatgcgcc 19200 ttgatctaacccacaaaacc tgcaaacaaa tcctgcaaac cgcaattcaa aaaacacgcg 19260 cctcaggcacgcatgaagaa aaagaagcgt ccaagtctgg ttcagatgat gggaatacgt 19320 caggccagtccgcgtaccaa agccggcgcc aagtggctgt cgaagatcaa gaaaggtgag 19380 acatgtatgtatcgctgctg ggctactccg accaggatcc gtccatatcc tggaaaacac 19440 aacccatccatccgaggggt tttgtagcta acagcgtgtc agcccaagtg taactcctaa 19500 ctttccttcaactcaactct tttctctgga acaattggct cgctctagct cgaaattatt 19560 tcctcaacctttcgcctttc cagtgcacaa aggtagaaac gccatggatc tctataaatc 19620 cgacattatattgaatttga ggtagaagtc gtgatctttg gcgtttgtac ctcagtgcat 19680 cttgctgtatagtggaatcc aaaagctaat gatattacct cgaattccca gactcagtgg 19740 ccctgtcgcaggtccatcta tcgcctctgg gatcacaggc gagcttctcg caggccaatc 19800 agaatcgcatcgagaacgtg gccgactggg aggcgattgc caaaaagtac cgggcactgg 19860 ttggcgacgaggagggtgga tcgctcaagg actcggatgc ggagagtgga tcgcaggagg 19920 gtagcggaggacaacagcca ccggcacagg tgggcagacg agccatcaag gccaccctgg 19980 cagacactacaaaatagaca cacagaaatg acacagaaaa aacagaaaaa cagcttcgga 20040 tgcttaattaactacgtttt gattgcaggt ctaagcttca tctatctctt caaactatcc 20100 ttcctgactatctctatctc ttctcgacta tccaagcgtc tgtccttctg taattctaag 20160 atctaactctaagaaactct atccgtaagc tgcaccttgg gtatggtttt ctcagactct 20220 ggaacccacttcttttggtt caactggagt atgggaaaat cagactaaaa tccttaagtt 20280 aagccttcactttctaaact aattttagct agaatattga aattgttttg agtaaccttt 20340 aaagcgaaagctgattgttt attttgatat gattttccgt tggagttttc tacgattagc 20400 gaaacaacaaaaaaaagttt tccatgttcg agatttttaa agtaagttaa ttcgtccttt 20460 ttggactcaatttgccttac attttttgaa accaactcct agcattttgt attaagctaa 20520 tgattgcgaccatatcgtta ataatgattg tcttagagat gttaagtaaa ttgaacttta 20580 gcttcaatcggagctaaaag tcaagcggtt ttatataaat ctcgcataat ctcattgttt 20640 tccggtaattgtcaagtaac aacgttcact ctacttacta agctttggtt cattttttat 20700 aacaaatgagcgcataaaat tgttaactgt acttgattgt aaataaataa gtcttatttt 20760 aaaatattgtactattgctt cagcttgtaa tcattgcata ctttttggcg gcactggcat 20820 ataccgccatctatcggagg aaacaaaatt ttaaaattat gtttagcatt attttttcta 20880 attaaactatttttgggttc atgcttataa tacaattata attttataat tataagtctg 20940 tatttttgaataaatggatt gtttttgtgt ttgttattta tatcgtacgt tactcgcgtg 21000 ctgccagattatcaaaaata gctctcgctt atttcccatt cacttgagcg acatctgtga 21060 atgaaatatagaacatgcgg ataaggtatt ttttggtttt cattaaattc cgctaggtgg 21120 cgaatgcaaatgtaaaatta atgtaaattg ataaatcatt gaaactaatg attaaaaaaa 21180 attgatttagaatttaataa tatatattgt attttgaata atatttccta aacctttcat 21240 ttaaataaaaatgattacga ttttatcata aatgttggtt tttattctaa cttagtaact 21300 gcaagctggtttgattatgc caagataatt tcaaaatagg ctagaattct ctcctttaaa 21360 ccatgtaatcatggccataa agctaagaac gggcaataaa attcgcttaa tttgcctgct 21420 gaattgacagattaccaaga ggcactcagg cgtcattagc cgggccagca gaaaagcgac 21480 agaaaccgcatcggaaattg accaaggtgt tgaacttcgg aattgcattt taatttggct 21540 tcaagctgcagtttgctgtt gttttcgcct cgattgcagg tgtcacagtc ggtttaaatg 21600 tgttgaaaacctcaagtggt caatgtttgc tgcttgctgc actcgcactc gtattattac 21660 acataattgccccttgccgt tgacattgtt gctgtgtggc agttgcactt gcatttgcag 21720 ttgctgctgtgcttgatatt tgccaccgat aaaatgcata catacatgca aaaatatatg 21780 aaaacgaaaagcaaacgagt ttctgtagcc gcagccaagg tttatggcca caagcgtgtc 21840 aatttaagctgcaattaggc agttaataaa tttaaccgat cttaccagtc agataccagg 21900 tccagatgccagctgattaa tgccactttc ccagcgattc ggtagctgca acgtacaaaa 21960 ctccaaatggattccaatcg gattcgatgc tggcgatgct gtggctgtcc gtcatccatc 22020 aaaggtttcttctacggacc aggaagcagt ttcgattcga ttcgatccgg gcttccatgg 22080 cttcagcctccgcgactcgg catcgtgcaa catgtgtgtg gtgtgttggc acagcaggtg 22140 acatttccaggccagatcag gaaaatgtaa ataaatgatc ggacattgga cgacacccat 22200 gcccataaccatacccatat gatcaacctg gctgaacacg acatggagca agttgtacct 22260 ggttatacgactatatgttg ctgttcatgt tgctgttgct ttgatataca aaacactttt 22320 tcatatcgaaatttgtgata ggccgtgatt aatggcgagc gacacaaaca cttaatttga 22380 cgccaggcccgtagctggcg ccttggggaa atggcagaga tccgaacgca aactctttgg 22440 gtgcacagagagaaaagatg ctaattttcc attaaaagta tttagtatca gcttgaatga 22500 taggtaggttactgttaaag cgtttctgtt gagctaatag gcattaataa atgccattga 22560 acaactaacatttaagacta ttttataagt aatgagatca taaatagtaa aaatgtagtt 22620 acctctttttttcatcctgt agctttgaat ttgctgctgg tttgctggct gggagaaaca 22680 ataatctcgggcaagattaa ttattgtaat cacatcaaca gcagagccat gcgaacggat 22740 tctcgtattcgtattcgttt tcgttttcgg aatgggagtc acagaaaaac caacacgaaa 22800 atgatcaatgatcatcgctg ggttctctgt tgatttttat agcgaacgcc cgatcgccgg 22860 cctgggttacacatttcatt ggctaatcaa gatgctaatt tgaagaagat taattcgtgt 22920 gcgagtttctgactgcctgc caggcaagcc cgaagattcg aagattataa tctgctaagc 22980 aagaggaaactgaaggctta ttattaatac aggccaacac agcccccaga aatgtgtctt 23040 gtatttaattaaatacgcgc acactgggaa aagcaattcc aatgaattct taatctattt 23100 tctaattttataggacatta aaaccatatc ttaaaataaa aactcttgta tcgaaatcat 23160 taaaatgttatgcttacttg caaagactta tcaccatttt tttcgcgtgt atctgccatt 23220 tagccacatcccagaaatgt ggagagtttc gggtgagtgt tggcttggca gtgcagtgac 23280 acgcagattaattgaaattt tatgagtagc gcagacgtaa acaatcagcg agaccacctt 23340 ttgccagccccttaggtcat aggagctcgc caagatcccc ctgctcggat ggcgtatcca 23400 tgtccagattccaagctcca gcttgactac actaactggc caagtcggca acggacagct 23460 gtggctcaccccgtggccaa aagaaacttg caacattatg aaaaatggac cacagccatg 23520 cacagtggttgacagcagac ccttgggatg tgtggaaatt atttggaagc aacagcaaat 23580 aattccagataatgcaatta attcgatact tatatattat attctatatg tattttagta 23640 ttttaaagaacttctgttga taccactgtg ccctgtgatc ctgctgacgc gatcgccacg 23700 ctaattgatagactgtgaaa ttatttaaca acggctggaa agtgagctcg gcgtggctgc 23760 ggctcgaaaggagcttccaa gcgtggccag atgggtcaga aggctttcga cccggccatc 23820 aagaccagggtcggcacatc tttttggtgg ctctggtccc tggccgctgg ccaatcatcc 23880 atccagtggaggatcgcgga cttacggcta agtgaaaagt gttaaaaagc acgactcacg 23940 gcgggcagttgtgtcggatt tgaagacaaa tgagcagcgt cttttgacat ttgcgaaatt 24000 taaaatgtcagccgaaaact ggtgggtcgt ccacccttga cgaaggtttc ggatgggagg 24060 tcccggttccatagcggatc gccacgcttt gccggataag tcgcggagaa tttaaattaa 24120 aactcaggtgaaaggttatt aattcgcaag tggaactggg gcgtagctcg gctcactgtt 24180 aatactcgaaatctccactc atttgggtta atgctgatgg cactttgaca gggatgatga 24240 tgatggggatatgacgaatg ccagcggcga tgatgccaaa taaaatggaa gtgacagagt 24300 tcagtgcgttggttttaatt aataagcata tttccagaga gctttctttt cagcaaag 24358 2 1704 PRTDrosophila melanogaster amino acid sequence derived from nompC genomicsequence 2 Arg Thr Pro Met His Leu Ala Ala Glu Asn Gly His Ala His ValIle 1 5 10 15 Glu Ile Leu Ala Asp Lys Phe Lys Ala Ser Ile Phe Glu ArgThr Lys 20 25 30 Asp Gly Ser Thr Leu Met His Ile Ala Ser Leu Asn Gly HisAla Glu 35 40 45 Cys Ala Thr Met Leu Phe Lys Lys Gly Val Tyr Leu His MetPro Asn 50 55 60 Lys Asp Gly Ala Arg Ser Ile His Thr Ala Ala Ala Tyr GlyHis Thr 65 70 75 80 Gly Ile Ile Asn Thr Leu Leu Gln Lys Gly Glu Lys ValAsp Val Thr 85 90 95 Thr Asn Asn Tyr Thr Ala Leu His Ile Ala Val Glu SerAla Lys Pro 100 105 110 Ala Val Val Glu Thr Leu Leu Gly Phe Gly Ala AspVal His Val Arg 115 120 125 Gly Gly Lys Leu Arg Glu Thr Pro Leu His IleAla Ala Arg Val Lys 130 135 140 Asp Gly Asp Arg Cys Ala Leu Met Leu LeuLys Ser Gly Ala Ser Pro 145 150 155 160 Asn Leu Thr Thr Asp Asp Cys LeuThr Pro Val His Val Ala Ala Arg 165 170 175 His Gly Asn Leu Ala Thr LeuMet Gln Leu Leu Glu Asp Glu Gly Asp 180 185 190 Pro Leu Tyr Lys Ser AsnThr Gly Glu Thr Pro Leu His Met Ala Cys 195 200 205 Arg Ala Cys His ProAsp Ile Val Arg His Leu Ile Glu Thr Val Lys 210 215 220 Glu Lys His GlyPro Asp Lys Ala Thr Thr Tyr Ile Asn Ser Val Asn 225 230 235 240 Glu AspGly Ala Thr Ala Leu His Tyr Thr Cys Gln Ile Thr Lys Glu 245 250 255 GluVal Lys Ile Pro Glu Ser Asp Lys Gln Ile Val Arg Met Leu Leu 260 265 270Glu Asn Gly Ala Asp Val Thr Leu Gln Thr Lys Thr Ala Leu Glu Thr 275 280285 Ala Phe His Tyr Cys Ala Val Ala Gly Asn Asn Asp Val Leu Met Glu 290295 300 Met Ile Ser His Met Asn Pro Thr Asp Ile Gln Lys Ala Met Asn Arg305 310 315 320 Gln Ser Ser Val Gly Trp Thr Pro Leu Leu Ile Ala Cys HisArg Gly 325 330 335 His Met Glu Leu Val Asn Asn Leu Leu Ala Asn His AlaArg Val Asp 340 345 350 Val Phe Asp Thr Glu Gly Arg Ser Ala Leu His LeuAla Ala Glu Arg 355 360 365 Gly Tyr Leu His Val Cys Asp Ala Leu Leu ThrAsn Lys Ala Phe Ile 370 375 380 Asn Ser Lys Ser Arg Val Gly Arg Thr AlaLeu His Leu Ala Ala Met 385 390 395 400 Asn Gly Phe Thr His Leu Val LysPhe Leu Ile Lys Asp His Asn Ala 405 410 415 Val Ile Asp Ile Leu Thr LeuArg Lys Gln Thr Pro Leu His Leu Ala 420 425 430 Ala Ala Ser Gly Gln MetGlu Val Cys Gln Leu Leu Leu Glu Leu Gly 435 440 445 Ala Asn Ile Asp AlaThr Asp Asp Leu Gly Gln Lys Pro Ile His Val 450 455 460 Ala Ala Gln AsnAsn Tyr Ser Glu Val Ala Lys Leu Phe Leu Gln Gln 465 470 475 480 His ProSer Leu Val Asn Ala Thr Ser Lys Asp Gly Asn Thr Cys Ala 485 490 495 HisIle Ala Ala Met Gln Gly Ser Val Lys Val Ile Glu Glu Leu Met 500 505 510Lys Phe Asp Arg Ser Gly Val Ile Ser Ala Arg Asn Lys Leu Thr Asp 515 520525 Ala Thr Pro Leu Gln Leu Ala Ala Glu Gly Gly His Ala Asp Val Val 530535 540 Lys Ala Leu Val Arg Ala Gly Ala Ser Cys Thr Glu Glu Asn Lys Ala545 550 555 560 Gly Phe Thr Ala Val His Leu Ala Ala Gln Asn Gly His GlyGln Val 565 570 575 Leu Asp Val Leu Lys Ser Thr Asn Ser Leu Arg Ile AsnSer Lys Lys 580 585 590 Leu Gly Leu Thr Pro Leu His Val Ala Ala Tyr TyrGly Gln Ala Asp 595 600 605 Thr Val Arg Glu Leu Leu Thr Ser Val Pro AlaThr Val Lys Ser Glu 610 615 620 Thr Pro Thr Gly Gln Ser Leu Phe Gly AspLeu Gly Thr Glu Ser Gly 625 630 635 640 Met Thr Pro Leu His Leu Ala AlaPhe Ser Gly Asn Glu Asn Val Val 645 650 655 Arg Leu Leu Leu Asn Ser AlaGly Val Gln Val Asp Ala Ala Thr Ile 660 665 670 Glu Asn Met His Gly HisIle Gln Met Val Glu Ile Leu Leu Gly Gln 675 680 685 Gly Ala Glu Ile AsnAla Thr Asp Arg Asn Gly Trp Thr Pro Leu His 690 695 700 Cys Ala Ala LysAla Gly His Leu Glu Val Val Lys Leu Leu Cys Glu 705 710 715 720 Ala GlyAla Ser Pro Lys Ser Glu Thr Asn Tyr Gly Cys Ala Ala Ile 725 730 735 TrpPhe Ala Ala Ser Glu Gly His Asn Glu Val Leu Arg Tyr Leu Met 740 745 750Asn Lys Glu His Asp Thr Tyr Gly Leu Met Glu Asp Lys Arg Phe Val 755 760765 Tyr Asn Leu Met Val Val Ser Lys Asn His Asn Asn Lys Pro Ile Gln 770775 780 Glu Phe Val Leu Val Ser Pro Ala Pro Val Asp Thr Ala Ala Lys Leu785 790 795 800 Ser Asn Ile Tyr Ile Val Leu Ser Thr Lys Lys Glu Arg AlaLys Asp 805 810 815 Leu Val Ala Ala Gly Lys Gln Cys Glu Ala Met Ala ThrGlu Leu Leu 820 825 830 Ala Leu Ala Ala Gly Ser Asp Ser Ala Gly Lys IleLeu Gln Ala Thr 835 840 845 Asp Lys Arg Asn Val Glu Phe Leu Asp Val LeuIle Glu Asn Glu Gln 850 855 860 Lys Glu Val Ile Ala His Thr Val Val GlnArg Tyr Leu Gln Glu Leu 865 870 875 880 Trp His Gly Ser Leu Thr Trp AlaSer Trp Lys Ile Leu Leu Leu Leu 885 890 895 Val Ala Phe Ile Val Cys ProPro Val Trp Ile Gly Phe Thr Phe Pro 900 905 910 Met Gly His Lys Phe AsnLys Val Pro Ile Ile Lys Phe Met Ser Tyr 915 920 925 Leu Thr Ser His IleTyr Leu Met Ile His Leu Ser Ile Val Gly Ile 930 935 940 Thr Pro Ile TyrPro Val Leu Arg Leu Ser Leu Val Pro Tyr Trp Tyr 945 950 955 960 Glu ValGly Leu Leu Ile Trp Leu Ser Gly Leu Leu Leu Phe Glu Leu 965 970 975 ThrAsn Pro Ser Asp Lys Ser Gly Leu Gly Ser Ile Lys Val Leu Val 980 985 990Leu Leu Leu Gly Met Ala Gly Val Gly Val His Val Ser Ala Phe Leu 995 10001005 Phe Val Ser Lys Glu Tyr Trp Pro Thr Leu Val Tyr Cys Arg Asn Gln1010 1015 1020 Cys Phe Ala Leu Ala Phe Leu Leu Ala Cys Val Gln Ile LeuAsp Phe 1025 1030 1035 1040 Leu Ser Phe His His Leu Phe Gly Pro Trp AlaIle Ile Ile Gly Asp 1045 1050 1055 Leu Leu Lys Asp Leu Ala Arg Phe LeuAla Val Leu Ala Ile Phe Val 1060 1065 1070 Phe Gly Phe Ser Met His IleVal Ala Leu Asn Gln Ser Phe Ala Asn 1075 1080 1085 Phe Ser Pro Glu AspLeu Arg Ser Phe Glu Lys Lys Asn Arg Asn Arg 1090 1095 1100 Gly Tyr PheSer Asp Met Glu Gln Met Thr Cys Pro His Pro Asp Leu 1105 1110 1115 1120Arg Arg Trp Arg Ile Met Ser Ile Val Ala Ser Ala Asn Ser Asp Glu 11251130 1135 Ser Thr Arg Thr Thr Phe Pro Gly Gly Thr Ser Thr Ser Pro HisSer 1140 1145 1150 Leu Leu Glu Ile Pro Ser Pro Cys Met His Val Asp ValPhe Ile Gln 1155 1160 1165 Ser Ile Gln Thr Lys Ile Lys Gln Ser Ile SerAsn Ile Asp Ile Thr 1170 1175 1180 Asn Ala Arg Leu Pro Gly Ala Phe SerLeu Arg Arg Leu Pro Thr Thr 1185 1190 1195 1200 Lys Phe Cys Thr Ile GluThr Ile Glu Thr Asp Arg Ile Glu Ser Ile 1205 1210 1215 Thr Lys Asn AspAsn Ala Thr Asp Thr Asp Tyr Arg Cys Ser Tyr Met 1220 1225 1230 Leu GlyPro Met Thr Pro Phe Leu Ala Phe Glu Arg Leu Phe Phe Ala 1235 1240 1245Val Phe Gly Gln Thr Thr Thr Leu Asp Ile Asn Pro Met Arg His Leu 12501255 1260 Arg Pro Glu Trp Thr Glu Val Leu Phe Lys Phe Val Phe Gly IleTyr 1265 1270 1275 1280 Leu Leu Val Ser Val Val Val Leu Ile Asn Leu LeuIle Ala Met Met 1285 1290 1295 Ser Asp Thr Tyr Gln Arg Ile Gln Met AsnArg Asn Trp Gly Leu Val 1300 1305 1310 Asp Arg Thr Asn Gln Arg Asn LysLys Lys Lys Lys Asn His Ile Ile 1315 1320 1325 Glu Ser Thr Asn Pro ThrTrp Ala Ser Val Ile Phe Leu Phe Phe Lys 1330 1335 1340 Ile Ile Ser ThrPro Ala Asn Ile Cys Val Leu Ser Gly Gly Val Tyr 1345 1350 1355 1360 LeuTyr Leu Tyr Leu Tyr Leu Glu Met Tyr Leu Trp Val Ser Asp Thr 1365 13701375 Val Arg Met His Pro Ile Asn Ser Phe Glu Leu Leu Phe Phe Ala Val1380 1385 1390 Phe Gly Gln Thr Thr Thr Glu Gln Thr Gln Val Asp Lys IleLys Asn 1395 1400 1405 Val Ala Thr Pro Thr Gln Pro Tyr Trp Val Glu TyrLeu Phe Lys Ile 1410 1415 1420 Val Phe Gly Ile Tyr Met Leu Val Ser ValVal Val Leu Ile Asn Leu 1425 1430 1435 1440 Leu Ile Ala Met Met Ser AspThr Tyr Gln Arg Ile Gln Ala Gln Ser 1445 1450 1455 Asp Ile Glu Trp LysPhe Gly Leu Ser Lys Leu Ile Arg Asn Met His 1460 1465 1470 Arg Thr ThrThr Ala Pro Ser Pro Leu Asn Leu Val Thr Thr Trp Phe 1475 1480 1485 MetTrp Ile Val Glu Lys Val Lys Val Lys Ser Gln Val Thr Lys Val 1490 14951500 Ala Phe Gln Pro Leu Ser Leu Cys Leu Ser Leu Ser Ile Arg Ile Leu1505 1510 1515 1520 Tyr Pro Val Ser Tyr Thr Cys Phe His Ile Cys Met LysLys Lys Lys 1525 1530 1535 Arg Pro Ser Leu Val Gln Met Met Gly Ile ArgGln Ala Ser Pro Arg 1540 1545 1550 Thr Lys Ala Gly Ala Lys Trp Leu SerLys Ile Lys Lys Ser Val Ala 1555 1560 1565 Leu Ser Gln Val His Leu SerPro Leu Gly Ser Gln Ala Ser Phe Ser 1570 1575 1580 Gln Ala Asn Gln AsnArg Ile Glu Asn Val Ala Asp Trp Glu Ala Ile 1585 1590 1595 1600 Ala LysLys Tyr Arg Ala Leu Val Gly Asp Glu Glu Gly Gly Ser Leu 1605 1610 1615Lys Asp Ser Asp Ala Glu Ser Gly Ser Gln Glu Gly Ser Gly Gly Gln 16201625 1630 Gln Pro Pro Ala Gln Val Gly Arg Arg Ala Ile Lys Ala Thr LeuAla 1635 1640 1645 Asp Thr Thr Lys Ser Lys Leu His Leu Ser Leu Gln ThrIle Leu Pro 1650 1655 1660 Asp Tyr Leu Tyr Leu Phe Ser Thr Ile Gln AlaSer Val Leu Leu Cys 1665 1670 1675 1680 Thr Leu Gly Met Val Phe Ser AspSer Gly Thr His Phe Phe Trp Phe 1685 1690 1695 Asn Trp Ser Met Gly LysSer Asp 1700 3 6156 DNA Drosophila melanogaster nompC cDNA sequence 3tttctcgtcg ctccgaaaaa aggcaaaata gtaggcaacc tgaaatccag agttgtagtt 60ggggactctt ttggccaaaa tacaaggagg agaaaaatag aaaataataa agggggcacc 120gccgttaacg cacacgcaac cgaagccata aaggggctaa acatataaat ttgtgtagta 180aaagtgaaga aagcgaaaga atcaaagtgg aataatagcg agtgtttttc ggtttgctag 240tgtgtttctg agtcggagtt tgtgtgtgtg tgtttgtgtg attcctagtg tgtctgttgc 300tgttgccaat gaaaatgcaa attgttggta acaaatattg gtaaaatgcg gaggccgtag 360gaatttgtgc aatgcgagtg cgaagtgaag gagcccgaaa ctatgcagct aaaaacccgc 420catcctaccc cgcatcgaat caataataat acaataaccc aaacgtatta cacggataat 480ggcagcataa accagttaac atccgacagt gtttccgcct aaccatcgag cacctagctc 540atcccccctg ccaccaaccc ttcgaaaaat ccccatgatc agcgccggat tgtggagcag 600taactagcga ggcataccag gatgtcgcag ccgcgcggag ggcgtggcgg tgggcgtggc 660ggcggagtgg gtcgcaaaac cccctcctcg ctgaccggcc caccggatga gtcggctacg 720cccagcgaac gggctacgcc cgccagcaaa gcagactccg atcccaagga cgatagctcg 780agcaatggcg acaagaagga tatggatctt tttccagccc caaagccgcc gagtgccggc 840gcctccattc gggacacggc gaacaaggtg ctcggattgg ccatgaaaag cgagtggacg 900cccatcgagg cggagctcaa gaagctggaa aagtatgtgg ccaatgtggg cgaggatggc 960aatcacatac cgctggccgg cgttcacgac atgaataccg gcatgacgcc gctgatgtac 1020gcaacgaagg acaataagac ggccataatg gatcgcatga ttgagctggg cgccgatgtg 1080ggagcccgca ataatgataa ttataatgtg ctacatattg ccgcaatgta ttcgcgtgag 1140gatgtcgtca aattgttgct aacaaaacgc ggcgtggatc ccttctccac cggtggctcg 1200cgttcgcaaa ctgcggtgca tttggtgtcc agtcgacaaa ccggaactgc aactaatatc 1260ctgcgcgctc tgctcgcggc agctggcaag gatattcgct tgaaagcgga cggccgtggc 1320aaaataccat tgctcctggc cgtggagtcg ggcaaccagt ccatgtgcag ggagctcctg 1380gctgcacaaa cagcagagca gctcaaggca acgacggcca atggagacac ggccttgcat 1440ttggccgcca gacggcggga cgtggacatg gtccgcatcc tggttgatta cggaacgaat 1500gtggacacgc agaatgggga gggccagacg ccacttcata tcgcggccgc cgaaggcgat 1560gaggctctac tcaagtactt ctatggcgtg cgcgcctcag cgtccattgc ggacaatcaa 1620gatcgcactc cgatgcactt ggccgccgag aatgggcacg cgcacgtcat cgagatactg 1680gccgacaagt tcaaggcgag catcttcgag cgcaccaagg atggcagcac gctgatgcac 1740attgcgtcac tcaacggtca tgctgagtgc gccacgatgc tcttcaagaa gggcgtctac 1800ctccatatgc ccaacaagga tggagcccgg agtattcaca ccgccgccgc ctatggtcac 1860acgggaatca tcaacaccct gctacagaag ggcgagaaag tggatgtgac caccaatgat 1920aactatacag cactgcacat agccgtggaa tcggctaagc ccgccgttgt ggaaaccctg 1980ctgggatttg gagcagatgt ccatgtccgt ggcggaaaac tacgtgagac cccgctgcac 2040attgcggcac gagtgaagga tggagatagg tgtgccctca tgttgctgaa gtcgggagcc 2100agtccaaatt tgaccacgga tgactgtctg acccccgtgc atgtggcggc tcgtcatggc 2160aatctggcca cgttgatgca actcctcgag gacgaaggag atccgctgta caaatcgaat 2220actggagaga caccgctgca catggcctgt cgtgcttgcc acccggatat tgtgcgtcat 2280ctcatcgaga cggtgaagga gaaacacggt ccggataagg ccaccaccta tataaactcg 2340gtaaacgagg acggcgccac ggcgttgcat tacacctgcc aaatcaccaa ggaggaggtt 2400aagattcccg aatccgacaa gcagatcgtt cggatgctcc tcgaaaatgg tgcggatgtc 2460acgttgcaaa cgaaaactgc cttggagacc gctttccact actgcgccgt ggccggcaac 2520aatgatgtgc tgatggagat gatctcacat atgaatccca cagacatcca aaaggccatg 2580aaccggcaat catcggtggg ctggactcca ctgctgattg cttgccatcg agggcacatg 2640gagctggtca ataatctact ggcgaatcac gctcgagtgg atgtcttcga tacggaagga 2700cgatctgcct tgcatttggc tgctgagcga ggatacctgc atgtgtgtga tgccctgctg 2760accaataagg cttttattaa ctccaagtcc cgcgtgggac gcactgcact acatctggca 2820gccatgaatg gatttacgca tctggtgaaa ttcctgatca aggatcacaa tgcagttatc 2880gatattctaa cgttgagaaa gcaaacgccg ctccatttgg cggcagccag cgggcagatg 2940gaagtctgtc agctgctcct cgagctgggc gccaatatcg atgcgacgga cgatctgggc 3000cagaagccaa tccacgtcgc cgcccagaac aactactctg aagtggccaa actcttcctg 3060cagcagcatc catccctggt gaatgccacc agcaaggatg gaaacacatg tgcccacatt 3120gccgccatgc agggatccgt caaggtgatc gaggagctga tgaagttcga tcgatcgggt 3180gtgatttcgg cgcggaataa acttacggat gccacgcccc ttcagctggc cgccgagggc 3240ggacatgcgg atgtggtgaa ggctcttgtg agagctggtg cctcctgcac cgaagagaac 3300aaggcgggat tcaccgccgt tcatctggcg gcacagaatg gacatggtca ggtcttggat 3360gtgctgaaaa gcacaaactc actaaggatc aatagcaaaa agttgggtct gacgccgctt 3420catgtggctg cctattacgg acaggcggat accgtgcggg aattgctgac cagtgttccc 3480gccaccgtca agtcggaaac tccaacggga caaagtttat ttggggatct gggcacggag 3540tccggaatga caccactaca cttggcggcc ttttccggca acgagaacgt ggtgcgactg 3600ctcctcaact ctgcgggtgt tcaagtggat gcggcgacca tcgagaacgg ctataatcca 3660ctccatttgg cttgcttcgg tggtcacatg tcagtggtcg gtttgctcct aagtcggtcg 3720gcggaactcc tccaatcgca ggatcgtaac ggcaggacgg gcctgcatat cgccgccatg 3780catggccaca tccagatggt ggagattctg ctcggccagg gcgcggagat caacgcaacc 3840gatcggaacg gttggacgcc actgcattgt gctgccaaag ctggccactt ggaggtggtg 3900aagttgctgt gcgaggcggg tgcctcgcca aaatcggaga ccaactacgg ttgcgccgcc 3960atttggttcg ccgcctccga gggacacaac gaggtcctgc ggtatctgat gaacaaggag 4020cacgacacct acggcctgat ggaggacaag cgattcgtgt acaacctgat ggtggtgtcc 4080aagaaccaca acaacaagcc cattcaggag tttgtcctgg tatcaccagc acccgtggat 4140acagccgcca aactgtccaa catctacata gtactctcga caaaggaaaa agagcgcgcc 4200aaggatctgg tagcagctgg caaacagtgc gaggcaatgg ccacggagct cttggccctg 4260gcagctgggt cagattccgc cggaaagatc cttcaagcca ccgataagcg aaacgtggag 4320tttctcgacg ttctcattga aaatgagcag aaggaagtga ttgcccacac ggtagttcag 4380cgatacttgc aagaactctg gcatggctcc ctgacgtggg catcctggaa aatccttctg 4440ctgctcgtgg ccttcatagt ctgcccacca gtgtggattg gattcacatt cccgatgggt 4500cacaagttca acaaggtgcc catcatcaag ttcatgtcgt acctaacctc tcacatttac 4560ctcatgatcc acctgagcat cgtgggcata acgcccattt acccagtgct ccgattgagt 4620ttggtgccct actggtacga ggtgggtctt ctcatctggc tgagtggatt gctccttttc 4680gagctgacga atccgtcaga taaatcggga ctgggatcga taaaggtgct cgtgctgctg 4740ctcggcatgg ccggagtggg tgtccatgtc tcagcatttc tattcgtctc caaggagtac 4800tggccaactt tggtgtattg tcgaaatcag tgcttcgcgt tggccttcct gctggcctgt 4860gtgcagatcc tcgacttttt gtccttccac cacctattcg gtccctgggc catcatcatt 4920ggggatctgc tgaaggatct ggctcggttt ttggccgtcc tggccatctt tgtgtttggc 4980ttttccatgc acattgtggc cctgaatcag agctttgcca atttctcacc ggaggatctg 5040cgcagcttcg agaagaagaa ccgaaataga ggctacttca gtgacgtgcg catgcatccg 5100attaactcgt tcgagttgtt gttcttcgcc gtgttcggac aaacgacgac cgagcaaacg 5160caagttgaca aaatcaaaaa tgtagccacg cccactcaac cgtattgggt tgagtacctg 5220ttcaaaattg tctttggcat ttacatgttg gtgtcggtgg ttgtgctcat taacctgctg 5280attgctatga tgtcagacac ctatcaacgc attcaggtag tattgctaaa tgcgctttta 5340tctaactcga ctctatttat taactcgtac tttaaccata agtatataaa tttcatattg 5400cattgtgtat taatcattct ctatttcagc ataagaagta aatttacata tgaagatgat 5460ttatatttct tagatatata atagcggtag ttaggaagtg agctgttttg ggaacatatt 5520gagaaaatag ttaattaatc tggagaactt ggcatgctct gtaaatccat caactgccca 5580gacttgcatc ttccaggttt tttcaggaaa ataatgttag caatctgagg gatacaattt 5640tgtgaaagtg tatctcaaag atggaagcct gccgccttct agtgtagtac agtgcagagt 5700agctttagtg gattagccgc cttgaagtgt gccctgcttt tgtgaccagt gttgagcgag 5760gccaaaccag aaagtgttgg ttaacgcatg cttacaaaac cttatatata gaaatcgttg 5820ctgcatgctt atatgtctgt gtttgtcatt gtctaggact taagtctgaa gagatacacc 5880aatatggtgg ttaggttttg tatggtaatt ttgtgattgc catccaaaac aggcctctga 5940atttgtgtat ttctattatt aacaacctga tttttgcagc tcttaagtta cgtattaaca 6000aagtaaaaac ctgtaaaatc cgaggcttct gttcacgaaa ctcatcccgt ttattccttt 6060gttcttgttc tctcctatat catgtctcat ccatccaaca tcgcgcacct cgctaaccaa 6120taataaactg aacaaaaaaa aaaaaaaaaa actcga 6156 4 1619 PRT Drosophilamelanogaster amino acid sequence derived from nompC cDNA sequence 4 MetSer Gln Pro Arg Gly Gly Arg Gly Gly Gly Arg Gly Gly Gly Val 1 5 10 15Gly Arg Lys Thr Pro Ser Ser Leu Thr Gly Pro Pro Asp Glu Ser Ala 20 25 30Thr Pro Ser Glu Arg Ala Thr Pro Ala Ser Lys Ala Asp Ser Asp Pro 35 40 45Lys Asp Asp Ser Ser Ser Asn Gly Asp Lys Lys Asp Met Asp Leu Phe 50 55 60Pro Ala Pro Lys Pro Pro Ser Ala Gly Ala Ser Ile Arg Asp Thr Ala 65 70 7580 Asn Lys Val Leu Gly Leu Ala Met Lys Ser Glu Trp Thr Pro Ile Glu 85 9095 Ala Glu Leu Lys Lys Leu Glu Lys Tyr Val Ala Asn Val Gly Glu Asp 100105 110 Gly Asn His Ile Pro Leu Ala Gly Val His Asp Met Asn Thr Gly Met115 120 125 Thr Pro Leu Met Tyr Ala Thr Lys Asp Asn Lys Thr Ala Ile MetAsp 130 135 140 Arg Met Ile Glu Leu Gly Ala Asp Val Gly Ala Arg Asn AsnAsp Asn 145 150 155 160 Tyr Asn Val Leu His Ile Ala Ala Met Tyr Ser ArgGlu Asp Val Val 165 170 175 Lys Leu Leu Leu Thr Lys Arg Gly Val Asp ProPhe Ser Thr Gly Gly 180 185 190 Ser Arg Ser Gln Thr Ala Val His Leu ValSer Ser Arg Gln Thr Gly 195 200 205 Thr Ala Thr Asn Ile Leu Arg Ala LeuLeu Ala Ala Ala Gly Lys Asp 210 215 220 Ile Arg Leu Lys Ala Asp Gly ArgGly Lys Ile Pro Leu Leu Leu Ala 225 230 235 240 Val Glu Ser Gly Asn GlnSer Met Cys Arg Glu Leu Leu Ala Ala Gln 245 250 255 Thr Ala Glu Gln LeuLys Ala Thr Thr Ala Asn Gly Asp Thr Ala Leu 260 265 270 His Leu Ala AlaArg Arg Arg Asp Val His Met Val Arg Ile Leu Val 275 280 285 Asp Tyr GlyThr Asn Val Asp Thr Gln Asn Gly Glu Gly Gln Thr Pro 290 295 300 Leu HisIle Ala Ala Ala Glu Gly Asp Glu Ala Leu Leu Lys Tyr Phe 305 310 315 320Tyr Gly Val Arg Ala Ser Ala Ser Ile Ala Asp Asn Gln Asp Arg Thr 325 330335 Pro Met His Leu Ala Ala Glu Asn Gly His Ala His Val Ile Glu Ile 340345 350 Leu Ala Asp Lys Phe Lys Ala Ser Ile Phe Glu Arg Thr Lys Asp Gly355 360 365 Ser Thr Leu Met His Ile Ala Ser Leu Asn Gly His Ala Glu CysAla 370 375 380 Thr Met Leu Phe Lys Lys Gly Val Tyr Leu His Met Pro AsnLys Asp 385 390 395 400 Gly Ala Arg Ser Ile His Thr Ala Ala Ala Tyr GlyHis Thr Gly Ile 405 410 415 Ile Asn Thr Leu Leu Gln Lys Gly Glu Lys ValAsp Val Thr Thr Asn 420 425 430 Asp Asn Tyr Thr Ala Leu His Ile Ala ValGlu Ser Ala Lys Pro Ala 435 440 445 Val Val Glu Thr Leu Leu Gly Phe GlyAla Asp Val His Val Arg Gly 450 455 460 Gly Lys Leu Arg Glu Thr Pro LeuHis Ile Ala Ala Arg Val Lys Asp 465 470 475 480 Gly Asp Arg Cys Ala LeuMet Leu Leu Lys Ser Gly Ala Ser Pro Asn 485 490 495 Leu Thr Thr Asp AspCys Leu Thr Pro Val His Val Ala Ala Arg His 500 505 510 Gly Asn Leu AlaThr Leu Met Gln Leu Leu Glu Asp Glu Gly Asp Pro 515 520 525 Leu Tyr LysSer Asn Thr Gly Glu Thr Pro Leu His Met Ala Cys Arg 530 535 540 Ala CysHis Pro Asp Ile Val Arg His Leu Ile Glu Thr Val Lys Glu 545 550 555 560Lys His Gly Pro Asp Lys Ala Thr Thr Tyr Ile Asn Ser Val Asn Glu 565 570575 Asp Gly Ala Thr Ala Leu His Tyr Thr Cys Gln Ile Thr Lys Glu Glu 580585 590 Val Lys Ile Pro Glu Ser Asp Lys Gln Ile Val Arg Met Leu Leu Glu595 600 605 Asn Gly Ala Asp Val Thr Leu Gln Thr Lys Thr Ala Leu Glu ThrAla 610 615 620 Phe His Tyr Cys Ala Val Ala Gly Asn Asn Asp Val Leu MetGlu Met 625 630 635 640 Ile Ser His Met Asn Pro Thr Asp Ile Gln Lys AlaMet Asn Arg Gln 645 650 655 Ser Ser Val Gly Trp Thr Pro Leu Leu Ile AlaCys His Arg Gly His 660 665 670 Met Glu Leu Val Asn Asn Leu Leu Ala AsnHis Ala Arg Val Asp Val 675 680 685 Phe Asp Thr Glu Gly Arg Ser Ala LeuHis Leu Ala Ala Glu Arg Gly 690 695 700 Tyr Leu His Val Cys Asp Ala LeuLeu Thr Asn Lys Ala Phe Ile Asn 705 710 715 720 Ser Lys Ser Arg Val GlyArg Thr Ala Leu His Leu Ala Ala Met Asn 725 730 735 Gly Phe Thr His LeuVal Lys Phe Leu Ile Lys Asp His Asn Ala Val 740 745 750 Ile Asp Ile LeuThr Leu Arg Lys Gln Thr Pro Leu His Leu Ala Ala 755 760 765 Ala Ser GlyGln Met Glu Val Cys Gln Leu Leu Leu Glu Leu Gly Ala 770 775 780 Asn IleAsp Ala Thr Asp Asp Leu Gly Gln Lys Pro Ile His Val Ala 785 790 795 800Ala Gln Asn Asn Tyr Ser Glu Val Ala Lys Leu Phe Leu Gln Gln His 805 810815 Pro Ser Leu Val Asn Ala Thr Ser Lys Asp Gly Asn Thr Cys Ala His 820825 830 Ile Ala Ala Met Gln Gly Ser Val Lys Val Ile Glu Glu Leu Met Lys835 840 845 Phe Asp Arg Ser Gly Val Ile Ser Ala Arg Asn Lys Leu Thr AspAla 850 855 860 Thr Pro Leu Gln Leu Ala Ala Glu Gly Gly His Ala Asp ValVal Lys 865 870 875 880 Ala Leu Val Arg Ala Gly Ala Ser Cys Thr Glu GluAsn Lys Ala Gly 885 890 895 Phe Thr Ala Val His Leu Ala Ala Gln Asn GlyHis Gly Gln Val Leu 900 905 910 Asp Val Leu Lys Ser Thr Asn Ser Leu ArgIle Asn Ser Lys Lys Leu 915 920 925 Gly Leu Thr Pro Leu His Val Ala AlaTyr Tyr Gly Gln Ala Asp Thr 930 935 940 Val Arg Glu Leu Leu Thr Ser ValPro Ala Thr Val Lys Ser Glu Thr 945 950 955 960 Pro Thr Gly Gln Ser LeuPhe Gly Asp Leu Gly Thr Glu Ser Gly Met 965 970 975 Thr Pro Leu His LeuAla Ala Phe Ser Gly Asn Glu Asn Val Val Arg 980 985 990 Leu Leu Leu AsnSer Ala Gly Val Gln Val Asp Ala Ala Thr Ile Glu 995 1000 1005 Asn GlyTyr Asn Pro Leu His Leu Ala Cys Phe Gly Gly His Met Ser 1010 1015 1020Val Val Gly Leu Leu Leu Ser Arg Ser Ala Glu Leu Leu Gln Ser Gln 10251030 1035 1040 Asp Arg Asn Gly Arg Thr Gly Leu His Ile Ala Ala Met HisGly His 1045 1050 1055 Ile Gln Met Val Glu Ile Leu Leu Gly Gln Gly AlaGlu Ile Asn Ala 1060 1065 1070 Thr Asp Arg Asn Gly Trp Thr Pro Leu HisCys Ala Ala Lys Ala Gly 1075 1080 1085 His Leu Glu Val Val Lys Leu LeuCys Glu Ala Gly Ala Ser Pro Lys 1090 1095 1100 Ser Glu Thr Asn Tyr GlyCys Ala Ala Ile Trp Phe Ala Ala Ser Glu 1105 1110 1115 1120 Gly His AsnGlu Val Leu Arg Tyr Leu Met Asn Lys Glu His Asp Thr 1125 1130 1135 TyrGly Leu Met Glu Asp Lys Arg Phe Val Tyr Asn Leu Met Val Val 1140 11451150 Ser Lys Asn His Asn Asn Lys Pro Ile Gln Glu Phe Val Leu Val Ser1155 1160 1165 Pro Ala Pro Val Asp Thr Ala Ala Lys Leu Ser Asn Ile TyrIle Val 1170 1175 1180 Leu Ser Thr Lys Glu Lys Glu Arg Ala Lys Asp LeuVal Ala Ala Gly 1185 1190 1195 1200 Lys Gln Cys Glu Ala Met Ala Thr GluLeu Leu Ala Leu Ala Ala Gly 1205 1210 1215 Ser Asp Ser Ala Gly Lys IleLeu Gln Ala Thr Asp Lys Arg Asn Val 1220 1225 1230 Glu Phe Leu Asp ValLeu Ile Glu Asn Glu Gln Lys Glu Val Ile Ala 1235 1240 1245 His Thr ValVal Gln Arg Tyr Leu Gln Glu Leu Trp His Gly Ser Leu 1250 1255 1260 ThrTrp Ala Ser Trp Lys Ile Leu Leu Leu Leu Val Ala Phe Ile Val 1265 12701275 1280 Cys Pro Pro Val Trp Ile Gly Phe Thr Phe Pro Met Gly His LysPhe 1285 1290 1295 Asn Lys Val Pro Ile Ile Lys Phe Met Ser Tyr Leu ThrSer His Ile 1300 1305 1310 Tyr Leu Met Ile His Leu Ser Ile Val Gly IleThr Pro Ile Tyr Pro 1315 1320 1325 Val Leu Arg Leu Ser Leu Val Pro TyrTrp Tyr Glu Val Gly Leu Leu 1330 1335 1340 Ile Trp Leu Ser Gly Leu LeuLeu Phe Glu Leu Thr Asn Pro Ser Asp 1345 1350 1355 1360 Lys Ser Gly LeuGly Ser Ile Lys Val Leu Val Leu Leu Leu Gly Met 1365 1370 1375 Ala GlyVal Gly Val His Val Ser Ala Phe Leu Phe Val Ser Lys Glu 1380 1385 1390Tyr Trp Pro Thr Leu Val Tyr Cys Arg Asn Gln Cys Phe Ala Leu Ala 13951400 1405 Phe Leu Leu Ala Cys Val Gln Ile Leu Asp Phe Leu Ser Phe HisHis 1410 1415 1420 Leu Phe Gly Pro Trp Ala Ile Ile Ile Gly Asp Leu LeuLys Asp Leu 1425 1430 1435 1440 Ala Arg Phe Leu Ala Val Leu Ala Ile PheVal Phe Gly Phe Ser Met 1445 1450 1455 His Ile Val Ala Leu Asn Gln SerPhe Ala Asn Phe Ser Pro Glu Asp 1460 1465 1470 Leu Arg Ser Phe Glu LysLys Asn Arg Asn Arg Gly Tyr Phe Ser Asp 1475 1480 1485 Val Arg Met HisPro Ile Asn Ser Phe Glu Leu Leu Phe Phe Ala Val 1490 1495 1500 Phe GlyGln Thr Thr Thr Glu Gln Thr Gln Val Asp Lys Ile Lys Asn 1505 1510 15151520 Val Ala Thr Pro Thr Gln Pro Tyr Trp Val Glu Tyr Leu Phe Lys Ile1525 1530 1535 Val Phe Gly Ile Tyr Met Leu Val Ser Val Val Val Leu IleAsn Leu 1540 1545 1550 Leu Ile Ala Met Met Ser Asp Thr Tyr Gln Arg IleGln Val Val Leu 1555 1560 1565 Leu Asn Ala Leu Leu Ser Asn Ser Thr LeuPhe Ile Asn Ser Tyr Phe 1570 1575 1580 Asn His Lys Tyr Ile Asn Phe IleLeu His Cys Val Leu Ile Ile Leu 1585 1590 1595 1600 Tyr Phe Ser Ile ArgSer Lys Phe Thr Tyr Glu Asp Asp Leu Tyr Phe 1605 1610 1615 Leu Asp Ile 59758 DNA Caenorhabditis elegans nompC genomic nucleotide sequence 5ctttgccgct taaaattttg cagtgacata tccttatgga acactttcaa atgacacatg 60tctcgtttta aagtctgacg gtaaactaaa aacatttcct tgtaagccta aacctaagcc 120aaagcctaag cctaataagc ctagctaacg ctcgccactg acgccaagcc taagactaat 180cctacgccaa tgcctaaaac tgacactgaa ataaaagtca aaagccaaaa gccaaaagcc 240aaaacctaag gccgaagcat aaggccaaag cctatgccta agcctgagcc tgagcttaaa 300tcctaagcct aagcctaagg ccaaagaaca agcctaagtc taagtccaag cctaagtatc 360aaaaacttac accgattccg ccaggctacc ctcagcacaa ttatcaactt tgttaacata 420tttatcggcg acggcgtggc gcttttctta ttcatctgtc tgatcagaat agctcttccg 480aacttccatt ccttatccga ctgtgcctga attcgttggt aggtgtcaga catcatagca 540atcagcaagt tgatcagcac aatcaaggtg accatcatgt agattccgaa tagaagtttt 600aagatgattt ttgcaaaatc tggaactaga tggagcgggg gcattgaatc gggctcgacg 660agtccgaaga gcgagaagaa gagcatttcg agggtttgag acggggaggc cagacgcatc 720agctcggcgc tgtcctcgtc gacaggctgg taggcaggct gaaaaaatct ctttcaaggc 780tcgtttttct tgcctaacct acctggaaga tactcgtcac gtggagtgtg aagcccgcca 840cgaacaacat caggatcaca aggaaacggg ccaaatcata cattagatcg ctgaaagctt 900cttcttctaa ggggtcagct caagccaagt actcaccgaa taatgatcgc ccagggaccg 960aacaaatgat gcactgtcag gaaatccagg tactctacaa aagcaaatag cagggcaaag 1020gcgaaaagtt gatttttcaa ataaagcatt gtccgggcga aatgtagctt ttcatcgtta 1080tccaggtggg ttaggaatac tgccgggagc aggaaggcta ggacatggac ggctatcgcc 1140atcgcggaaa ggactaggat taggaccttt acgattccta ggccagatcc tccaccgaca 1200gtggagagtt cggagaccag atttccagag agccagagca acaggagcca ttccacaggg 1260tttggaacca ccgaagttac ttcgtacctg gaaattgaga ttttgcaggt ctatctgata 1320tcccctaaat aaaatttaaa aaaataactt acatcttatg tgtaatattc aacaccacaa 1380ttgtcagcag tatcgtaaaa tagacatgag acacgatatg gcacacaaat ttaataatcg 1440gagctcttcc gatccgacta tccagtggaa gtgagaagta gaaccatgcc ggggggcata 1500ttagcacgaa gagggagaat gcgacaaact ttccgaatga ccagtcgaca cgggcagtcc 1560atacttctgt caggtagcgt tggacagacg cgtaggagac tacttctttc tggaaacggg 1620gtcgttgagg gttgactggt taggttaagc ttggagtgtt acctgctcat tttcaatgag 1680aacatctagt aggggccggc ctcgattgtc cttagccttc aggagaagag cggcattgta 1740ttcggtggcg gtgatccctg aaataatcta ggactagtaa attgtaagtc attttctgaa 1800aagattaaat agctaagtgg acctgtagcc ttggccggta actttggtcc aataaccttg 1860gtccagtaac cttaatcctg taaaccttgg tcctgaaatc ttggcctagt aacctaaaac 1920cttggtcctg tggtcctgac cctgttcctg tatccttggt tgggaaaccc tagtccttgt 1980cctggtttgg aaaccctggc ccggtagcct tggtccaggt actggtcctg tgcccttggt 2040cctggttctg gtcttggtcc cgaaaccttg gtccggcagt tttggttctg gtaccttggt 2100cgtgtaacct taaacccagt aaccttggac cggtaacctt ggtacagtaa ctttggtccg 2160gaagccctgg ctcggtaact ctggtcctgg tcatggtgtt ggtcctggcc cggacaccct 2220ggtccggtaa ccctggtcta gcaaccttgg tcttgaccta acaaccttgg ttctgtaacc 2280ttggtcttgt aacttcggcc ctgtatcctt ggcccaaaga ccttggtccg acagccttgg 2340ttctgatacc ttggtccagt aactttggtc gtggtcctgg ttcaggtcca gtaaccttga 2400cccgataatc ctggtcttac ctagtgacct tggcccggta atcctgatcc tggcccagta 2460accttggtcc agtacggtgg ccctgcaact atggcctagt agctttggtc cagtagccct 2520gatcccgaaa ccttggttca gtaaccttgg tcttggtcca gtaactttgg tctagtaacc 2580atagtccagt aaccctggtc ctgtaacctt ggtccgctag ccccgttagt catgttcccg 2640ctcctggtcc ggcagcattg gtccggtaat tttggacctc ccctgggcct tggcccaggg 2700catgttcctg gtccaggggg ccattttctt cgtttttcat tacctaccta acaactccac 2760agccatattc tcactgaaca ctgccacatt caacagatcc ttcgccctct ccttctcctt 2820ctccgacata tctctgtaca acgcggacaa cttgactgcc gtctcaattg gagcaggtga 2880ttgaagaata aactcttgta gaggctcatt gtcattggtt ttaccacaaa ccatcaagtc 2940gaatatgaac ttccgatctt ccatcaattg atgtgtgtca tgcttctgtt tcaggaggaa 3000tcgaagacat tctatatgat tatgagctgc agcaaagcac aatggaactt tgccctcctt 3060ggtctccgcc aatggatccg ctgaactatc gatgaacagc ttgacgacac tcaggtgccc 3120ggcacgagtg gcaaagtgaa gaccagtcca gccattctga tccatgacat tgatgttaga 3180tccctgagca atgagaagtg agaccatctc gtagtggcca ttctgagcgg ctaggtggag 3240cggggtcctg cctctccaat ccttggcgtg ctgctgctga gtagatctgg acaggagcat 3300tcctaccact gcgatgtggc cttgctgggc agccagatgg agggggatca cgttctgaaa 3360cggaatttta aacggggtca ctgaaaattt caagttacca ttgtagtact ggtcgcgtca 3420acttgcactc cctgattcag aagcatccgc acaagactgt cgtgtccact atgagcggct 3480aaatggagag gtgtgaagcc gtattcagtt gagaattcct tattgacatg gtgattgtag 3540atgggcggct cggaacggac tgttgcttgt acgtgcttga gcatttcatt gacgaaatcc 3600gaatttccgt agaacgcagc gatgtggaga gcgttgagac cggtctggaa atgctaggtt 3660cagggggaat cgagtttttt ttcagtacaa aattcataaa atttaaggct agctgtgaaa 3720aattgtgcta ccaaagtata ggccacggct tcaaatttga caggacttat tccactttgc 3780agatcagacc tttatgcatg aactgtactg ccacgtattg gaaaatgtta tttttgacag 3840ccttaccttt ctcgaacacc gtttccatag gatcttatcg aatgcctcca aaatcgatat 3900gaatccgttt ttggcgccaa ggtggagagc agtcattccg tgctgaaaat caattctgcc 3960taaaaatcgg taaaagaacc cctaccgaat tctcatcttc cgcgtttgct ccattctcca 4020gcagaatctt cacaatgttc gcgtgacctc ccgcagctgc catatgaagt gtagtggctt 4080ccagtgtttt ggtctttgcc tggattacca taggcttgtc gatcatcata agctcacgga 4140ccacggctag ggaaccctgg aacaatatta ttttagttgc aatcaaaagc tgaagcttcc 4200acccctacct tcatcgcagc aatatgtgcg caggtgaatc cattatgatc aattgcggtc 4260aacacactcc ggttgttatt tctcattttc aggaagagct tcacaacgtc ggggaagtca 4320ttctcagctg ccagatggag aggggtttga cccttgtcgt cacgtgcatt ggggtttgct 4380ccgagagcca gaagggtttg actcacagct agctgaccga attttgcggc aaagtggagg 4440gctgtctgga aattatttgt gtttctaatc aggagcttgc cgacaaattt gctcgaaccc 4500cgtattagaa actacgcaga accctgtctg ggcagtagat tacctctagc ttggatacta 4560tcttacctga ttatccagcg taattgcctc cagcgctgca ccatgatcct gcaccaggac 4620attcaccacc ttcacatgac catgctgagc tgctaagtgg agcggtgcct ctccggtttt 4680cgatttactg ttcacgaatg ctttgtgctg cagaagaagg tgaaccaggg agagatgccc 4740attgaaagct gccaggtgca gagcagtacg gcccatttca tcgaatacat caatacgggc 4800gtggtgctga aaaagtatga ggtatccggt ttgtgagaaa tcagtggtcc cccagtagcc 4860ttggcacagt aaccttggtc ctggtcctgg tcctcgccca gtaaccctgg tcctgtaacc 4920ctggtcctgt agccctggcc ctggtcctgg tcctggtcct ggcccagtaa ccttggtact 4980gtaaccatgg tactgtaacc ctggccctgg tcctggtcca gtaacctcgg ccctgtaacc 5040ttggtcctgg tcctggtcct ggtcctacac acaaaaccag taccttcaac aaaatattcg 5100ccactccaga atgccctctg gcacatgctt ccaacagcgg tgaccatccg ttcttgctct 5160gcttgttctg cacgatttgc accgcaccgg ctccgatctt attgaccatc gccaggagta 5220cagcttgatt tccggatctt gccgccatat gcatcgccgt ctcatttgca ttgagtgatg 5280gcatttctac cattccaccg tagtcgatca gaagatttac tagcttggca tcttctcctg 5340gaaagtgtaa ctggcgctgc tcgatttcag cggcgtagtg aagagctgtg aagccgtcct 5400gaaaaattta acttgaagct tcctgagatc cagagaaaga agctcacatt ggttctatga 5460ttgacatgtt ccttaagctg ttcttgggtc agaacttccg aaaggtgctt caaaatcatt 5520gatgctgctt caaaattgca tgacttggcg gccacctgga ggggtgtctc tccgatcttt 5580gagcttattt tcgagtcggc gttctcgtca agcaggagcc tggaaaaaag gaggttcttg 5640ggcttttaca ggatccgaca gaaaatagat ttctcgaact ttttcccgtt ttcgtactgt 5700caatttacca aatttcaagg taccctgttt ttataagtgc ttagaaattt caaaaatttc 5760aaaaattgtg ataaactggg gcgctgaatc cagaattggc acagaaattc agagtttctc 5820aattttcaaa gaggcttgta tgcaatgctt agaaatccta aattttgagc acgcagttca 5880cgggctccag gaccaagtgc acaataatct caaaattttt gggtcccaca gcagttgcgc 5940gctagctgaa aaattctgca cggcatgaga agtggcacct gtacgcaatt tgtctaccgt 6000atacctggac gtttagtagc gtttttttca aaattttttg gaccaaagct tttttcctca 6060aaacgcgcct aaacgtggct aaactgcaat tatcagttga gcgcgtttac actgatatac 6120actttgcagg gccgtgtgct gattggctct aaagtcggcg tggctaagca ctgattagtc 6180aagatcacct acttacctca tgatatcctt attcccactc ctggcagcaa tatgcagaca 6240agtctcccca tccatttgtg caacatccgg ctgccctcca cttttcagca acatcatcgc 6300acaatcccga ctctcggctc cattcaagct tgccgcaatg tgcagtgcag tttgtcctaa 6360aaccaatctt ccatgaaatc ttattaatct cttattaatt taatacctag ttccccgccc 6420ttcacatgaa tgtctgcacc acttcccagc agggtctcta caaccgaagc cttgccagat 6480tgaaccgcta cgtggagagc ggtgtagttg tctcgtgtac ggacatctac attagtaccc 6540cgagcaatga gcattttgac gacgtcgttg aagccagcag ctgctgcgga gtgaagaccc 6600agggctcctt ttttgttggg catgaagagg gggactcctg gaagttagaa ttaacaatgt 6660aagtcgaggg ggtgctgaga ccctgtaaac ctacctctct tcaaaaacgc caatgcggtg 6720ctagtatgtc ctgaacatgc ggcaatatgc agaagcgtcg acccatcacg ggtcctagcg 6780cgaattgagc caccaaactt gtcaattagt gactcgacca tcgaagtgtc acctcgctcc 6840gctgcaacgt gtaccggagt cttgtcctcc ttatcatgga tgttggcgtc ggcgcggagt 6900ttgaacatga tttttagcat attttgatct ccgacttcgg ctacctggaa aattggagat 6960agagatactg tatgtgtgca gaggcataaa ttcagatagg agtagtacca agctttgatg 7020gagcatgaat ctagttaagg tgtatcaggg atactgtaaa ggtacggtag tccggcatat 7080tgtatttctg acaaatctac tgtattgggt acagtaagct cagtaaccct tctgtgtacc 7140cgttacagtg aggcaagcta aacttaggcc atttttcctg ttaaaaaacc catttaaatg 7200ttgcctagat cagaacaagc ctcgaatttt acagcttcat cagcaaaatt tcagcttcag 7260gagctactta aagtttcaat ttccaccctt taacctacct catgtagcgg cgtccttccc 7320accctattct gcacattcgc attatcacat ccagccgcaa tcgctgtccg aaccgcttcg 7380atattcccac tccgagcggc caaatgaagc aaggtatccc cgtttccatc agctttcctg 7440gtttgttcat ccgaaggccc acttagcaga agctccacaa tattaacatt cccaaacttg 7500aatgccaagt gtatcggcaa ggatccatcc ccatcctctg ccattctttg atcagtatct 7560tccaaaatcc gcttcacaat tggaaatgct ttcttggatt ttctctcgca agccacatgg 7620attgccagct gctttttagg ccccgcacct tttcggagca gctcagagta tcgcttgagg 7680ataagctcaa gagtttcaac tccggagtac atggcggcaa tatgagtcgc gttacggcca 7740tctttagtgc tatagtccac tcgagcacct tttcggatca tcttgtctac gatttgatcc 7800ttgccagctt tgacggctag gaggaaggcg gtgaagccgt gctgcaagga gaatttttag 7860aaaatggcgg gtacaatcta aagtgaaaat ctaagtcagt ttcggggaat tttgggttag 7920ggctgctaaa cggctgcgag gggctcagca cattgaaaaa cgcagtgcta tatgtagttg 7980ttttgcagcc ccggggttcc gcaggcctca cgccactagc caccatggtc ctatgtatag 8040tgccgtgcgg aaccccgaaa gtgtcggcgg ctgccaaaca tctgcctatt gcactgcatt 8100gtccaatgcg aaggctcaac cccactgaag gtactacccc ctaatagtca gcagccctaa 8160tttgggtcaa accctaaaat tgcgaacttc accgacttgt ccgagttaca gcggaaaaaa 8220cttacattat cagccatact aaaatcactc cgcttgatag tctctatctc agactccaca 8280ttcgcccact catctctctt cgcgaaatac aaaatcttcg tctgaggatc cgccattgcc 8340aagtcctcac tcgacatttc ctcatgagac gatgcgtggg aggtgagact ctctcgaaac 8400agaggtttcc cgagaagacg atccggcggg gtgactgaat cacgggatgg ttgtttcgga 8460acgaagatga tccgtgagtt ctttccgatt tggagatggg tcgaggatcg gcggaggggt 8520ggtcggtcag ttgggatggt gtcggtggtg aggaggtcct ggaaagtggg tagaattagt 8580tttcgtaagc ttccaggcgt gcctacacgc cttcctgttg cctacgaaaa gtcctgaatc 8640taaaaagcat ttttggcagc atccatctaa aaaaatcggt atctttgagt agttttaaac 8700agtgttcttc cacgaaaaaa gttttccacg tcttgcctaa gtaagcctaa gcctcagctt 8760aagcctaagc atatgcctaa gcctaaatct aagcctaagc ctgagtctga gcctgagcct 8820aagcctattc caaagcttaa accgaagctt aagtctaggc cttagcctaa acctaagcct 8880aaacctaagc ctaagcctaa gcctaagcct caacctaagc ctaaacctaa acctaatcaa 8940atgcctacct ttttcccggt aaaccactcg gcccgtgtca ccgacgtcga gcgggtttcc 9000cgtttccgca cagttagaca tttttccgat cttgacattt tcagtattac cagaacagaa 9060aaagaaggga aaataataca tttctctcaa ctaattgggg ggcggacgca catggtgtcc 9120tccaacccat aaaaaagtac gaatgtgggc gattaattgc gaaaaatgcg cgaaatttat 9180ttacgactga cgacgagaag cattaaactt ttggtaaagg gtgctgtggg ggtactttgg 9240tgaaaatata gctaaaattt aggcttgggc ttgggcttag gcttaggctt aggtttcagc 9300tcaggcttag gcttcggctc aggctttggc gtaggcttaa actttggctt aggtttaagc 9360ttaggcttag gcttaggctt agtcttaggc ttaggcttag gcttaggctt aggctcaggt 9420ttaagcttag acttaggctc aggtttaggc ttggcgtcag tggcgagcgt tactgaagtg 9480atatttaatc actctgatga tatttaattc cgatgattaa tccacttttc tttttctcac 9540atttatgaac caagttctaa attaaggtgg gatattttaa ggtgtgttaa catatgatat 9600ttatttttta atttaaatat agtttctctt tttgcttctt tttataagtt ttgttaatga 9660acgcatagtt tacaaccgcc tcgctcaaat gtattttgat aaaagtgcgc tattaggctt 9720aagcgtcgcc ataccgccgg tgtggtcata aggaattc 9758 6 1709 PRT Caenorhabditiselegans amino acid sequence derived from nompC genomic sequence 6 MetSer Arg Ser Glu Lys Cys Leu Thr Val Arg Lys Arg Glu Thr Arg 1 5 10 15Ser Thr Ser Val Thr Arg Ala Glu Trp Phe Thr Gly Lys Lys Met Asp 20 25 30Ala Ala Lys Asn Ala Phe Asp Leu Leu Thr Thr Asp Thr Ile Pro Thr 35 40 45Asp Arg Pro Pro Leu Arg Arg Ser Ser Thr His Leu Gln Ile Gly Lys 50 55 60Asn Ser Arg Ile Ile Phe Val Pro Lys Gln Pro Ser Arg Asp Ser Val 65 70 7580 Thr Pro Pro Asp Arg Leu Leu Gly Lys Pro Leu Phe Arg Glu Ser Leu 85 9095 Thr Ser His Ala Ser Ser His Glu Glu Met Ser Ser Glu Asp Leu Ala 100105 110 Met Ala Asp Pro Gln Thr Lys Ile Leu Tyr Phe Ala Lys Arg Asp Glu115 120 125 Trp Ala Asn Val Glu Ser Glu Ile Glu Thr Ile Lys Arg Ser AspPhe 130 135 140 Ser Met Ala Asp Asn His Gly Phe Thr Ala Phe Leu Leu AlaVal Lys 145 150 155 160 Ala Gly Lys Asp Gln Ile Val Asp Lys Met Ile ArgLys Gly Ala Arg 165 170 175 Val Asp Tyr Ser Thr Lys Asp Gly Arg Asn AlaThr His Ile Ala Ala 180 185 190 Met Tyr Ser Gly Val Glu Thr Leu Glu LeuIle Leu Lys Arg Tyr Ser 195 200 205 Glu Leu Leu Arg Lys Gly Ala Gly ProLys Lys Gln Leu Ala Ile His 210 215 220 Val Ala Cys Glu Arg Lys Ser LysLys Ala Phe Pro Ile Val Lys Arg 225 230 235 240 Ile Leu Glu Asp Thr AspGln Arg Met Ala Glu Asp Gly Asp Gly Ser 245 250 255 Leu Pro Ile His LeuAla Phe Lys Phe Gly Asn Val Asn Ile Val Glu 260 265 270 Leu Leu Leu SerGly Pro Ser Asp Glu Gln Thr Arg Lys Ala Asp Gly 275 280 285 Asn Gly AspThr Leu Leu His Leu Ala Ala Arg Ser Gly Asn Ile Glu 290 295 300 Ala ValArg Thr Ala Ile Ala Ala Gly Cys Asp Asn Ala Asn Val Gln 305 310 315 320Asn Arg Val Gly Arg Thr Pro Leu His Glu Cys Leu Thr Val Thr Gly 325 330335 Thr Gln Lys Gly Tyr Val Ala Glu Val Gly Asp Gln Asn Met Leu Lys 340345 350 Ile Met Phe Lys Leu Arg Ala Asp Ala Asn Ile His Asp Lys Glu Asp355 360 365 Lys Thr Pro Val His Val Ala Ala Glu Arg Gly Asp Thr Ser MetVal 370 375 380 Glu Ser Leu Ile Asp Lys Phe Gly Gly Ser Ile Arg Ala ArgThr Arg 385 390 395 400 Asp Gly Ser Thr Leu Leu His Ile Ala Ala Cys SerGly His Thr Ser 405 410 415 Thr Ala Leu Ala Phe Leu Lys Arg Val Pro LeuPhe Met Pro Asn Lys 420 425 430 Lys Gly Ala Leu Gly Leu His Ser Ala AlaAla Ala Gly Phe Asn Asp 435 440 445 Val Val Lys Met Leu Ile Ala Arg GlyThr Asn Val Asp Val Arg Thr 450 455 460 Arg Asp Asn Tyr Thr Ala Leu HisVal Ala Val Gln Ser Gly Lys Ala 465 470 475 480 Ser Val Val Glu Thr LeuLeu Gly Ser Gly Ala Asp Ile His Val Lys 485 490 495 Gly Gly Glu Leu MetAsp Gly Glu Thr Cys Leu His Ile Ala Ala Arg 500 505 510 Ser Gly Asn LysAsp Ile Met Leu Leu Leu Asp Glu Asn Ala Asp Ser 515 520 525 Lys Ile SerSer Lys Ile Gly Glu Thr Pro Leu Gln Val Ala Ala Lys 530 535 540 Ser CysAsn Phe Glu Ala Ala Ser Met Ile Leu Lys His Leu Ser Glu 545 550 555 560Val Leu Thr Gln Glu Gln Leu Lys Glu His Val Asn His Arg Thr Asn 565 570575 Asp Gly Phe Thr Ala Leu His Tyr Ala Ala Glu Ile Glu Gln Arg Gln 580585 590 Leu His Phe Pro Gly Glu Asp Ala Lys Leu Val Asn Leu Leu Ile Asp595 600 605 Tyr Gly Gly Met Val Glu Met Pro Ser Leu Asn Ala Asn Glu ThrAla 610 615 620 Met His Met Ala Ala Arg Ser Gly Asn Gln Ala Val Leu LeuAla Met 625 630 635 640 Val Asn Lys Ile Gly Ala Gly Ala Val Gln Ile ValGln Asn Lys Gln 645 650 655 Ser Lys Asn Gly Trp Ser Pro Leu Leu Glu AlaCys Ala Arg Gly His 660 665 670 Ser Gly Val Ala Asn Ile Leu Leu Lys ValLeu Val Leu Cys Val Gly 675 680 685 Pro Gly Pro Gly Pro Gly Pro Arg LeuGln Gly Arg Gly Tyr Trp Thr 690 695 700 Arg Thr Arg Ala Arg Val Thr ValPro Trp Leu Gln Tyr Gln Gly Tyr 705 710 715 720 Trp Ala Arg Thr Arg ThrArg Thr Arg Ala Arg Ala Thr Gly Pro Gly 725 730 735 Leu Gln Asp Gln GlyTyr Trp Ala Arg Thr Arg Thr Arg Thr Lys Val 740 745 750 Thr Val Pro ArgLeu Leu Gly Asp His His Ala Arg Ile Asp Val Phe 755 760 765 Asp Glu MetGly Arg Thr Ala Leu His Leu Ala Ala Phe Asn Gly His 770 775 780 Leu SerLeu Val His Leu Leu Leu Gln His Lys Ala Phe Val Asn Ser 785 790 795 800Lys Ser Lys Thr Gly Glu Ala Pro Leu His Leu Ala Ala Gln His Gly 805 810815 His Val Lys Val Val Asn Val Leu Val Gln Asp His Gly Ala Ala Leu 820825 830 Glu Ala Ile Thr Leu Asp Asn Gln Thr Ala Leu His Phe Ala Ala Lys835 840 845 Phe Gly Gln Leu Ala Val Ser Gln Thr Leu Leu Ala Leu Gly AlaAsn 850 855 860 Pro Asn Ala Arg Asp Asp Lys Gly Gln Thr Pro Leu His LeuAla Ala 865 870 875 880 Glu Asn Asp Phe Pro Asp Val Val Lys Leu Phe LeuLys Met Arg Asn 885 890 895 Asn Asn Arg Ser Val Leu Thr Ala Ile Asp HisAsn Gly Phe Thr Cys 900 905 910 Ala His Ile Ala Ala Met Lys Gly Ser LeuAla Val Val Arg Glu Leu 915 920 925 Met Met Ile Asp Lys Pro Met Val IleGln Ala Lys Thr Lys Thr Leu 930 935 940 Glu Ala Thr Thr Leu His Met AlaAla Ala Gly Gly His Ala Asn Ile 945 950 955 960 Val Lys Ile Leu Leu GluAsn Gly Ala Asn Ala Glu Asp Glu Asn Ser 965 970 975 Gly Met Thr Ala LeuHis Leu Gly Ala Lys Asn Gly Phe Ile Ser Ile 980 985 990 Leu Glu Ala PheAsp Lys Ile Leu Trp Lys Arg Cys Ser Arg Lys Thr 995 1000 1005 Gly LeuAsn Ala Leu His Ile Ala Ala Phe Tyr Gly Asn Ser Asp Phe 1010 1015 1020Val Asn Glu Met Leu Lys His Val Gln Ala Thr Val Arg Ser Glu Pro 10251030 1035 1040 Pro Ile Tyr Asn His His Val Asn Lys Glu Phe Ser Thr GluTyr Gly 1045 1050 1055 Phe Thr Pro Leu His Leu Ala Ala His Ser Gly HisAsp Ser Leu Val 1060 1065 1070 Arg Met Leu Leu Asn Gln Gly Val Gln ValAsp Ala Thr Ser Thr Thr 1075 1080 1085 Met Met Ser Glu Lys Glu Lys GluArg Ala Lys Asp Leu Leu Asn Val 1090 1095 1100 Ala Val Phe Ser Glu AsnMet Ala Val Glu Leu Leu Ile Thr Ala Thr 1105 1110 1115 1120 Glu Tyr AsnAla Ala Leu Leu Leu Lys Ala Lys Asp Asn Arg Gly Arg 1125 1130 1135 ProLeu Leu Asp Val Leu Ile Glu Asn Glu Gln Lys Glu Val Val Ser 1140 11451150 Tyr Ala Ser Val Gln Arg Tyr Leu Thr Glu Val Trp Thr Ala Arg Val1155 1160 1165 Asp Trp Ser Phe Gly Lys Phe Val Ala Phe Ser Leu Phe ValLeu Ile 1170 1175 1180 Cys Pro Pro Ala Trp Phe Tyr Phe Ser Leu Pro LeuAsp Ser Arg Ile 1185 1190 1195 1200 Gly Arg Ala Pro Ile Ile Lys Phe ValCys His Ile Val Ser His Val 1205 1210 1215 Tyr Phe Thr Ile Leu Leu ThrIle Val Val Leu Asn Ile Thr His Lys 1220 1225 1230 Tyr Glu Val Thr SerVal Val Pro Asn Pro Val Glu Trp Leu Leu Leu 1235 1240 1245 Leu Trp LeuSer Gly Asn Leu Val Ser Glu Leu Ser Thr Val Gly Gly 1250 1255 1260 GlySer Gly Leu Gly Ile Val Lys Val Leu Ile Leu Val Leu Ser Ala 1265 12701275 1280 Met Ala Ile Ala Val His Val Leu Ala Phe Leu Leu Pro Ala ValPhe 1285 1290 1295 Leu Thr His Leu Asp Asn Asp Glu Lys Leu His Phe AlaArg Thr Met 1300 1305 1310 Leu Tyr Leu Lys Asn Gln Leu Phe Ala Phe AlaLeu Leu Phe Ala Phe 1315 1320 1325 Val Glu Tyr Leu Asp Phe Leu Thr ValHis His Leu Phe Gly Pro Trp 1330 1335 1340 Ala Ile Ile Ile Met Tyr AspLeu Ala Arg Phe Leu Val Ile Leu Met 1345 1350 1355 1360 Leu Phe Val AlaGly Phe Thr Leu His Val Thr Ser Ile Phe Gln Pro 1365 1370 1375 Ala TyrGln Pro Val Asp Glu Asp Ser Ala Glu Leu Met Arg Leu Ala 1380 1385 1390Ser Pro Ser Gln Thr Leu Glu Met Leu Phe Phe Ser Leu Phe Gly Leu 13951400 1405 Val Glu Pro Asp Ser Met Pro Pro Leu His Leu Val Pro Asp PheAla 1410 1415 1420 Lys Ile Ile Leu Lys Leu Leu Phe Gly Ile Tyr Met MetVal Thr Leu 1425 1430 1435 1440 Ile Val Leu Ile Asn Leu Leu Ile Ala MetMet Ser Asp Thr Tyr Gln 1445 1450 1455 Arg Ile Gln Ala Gln Ser Asp LysGlu Trp Lys Phe Gly Arg Ala Ile 1460 1465 1470 Leu Ile Arg Gln Met AsnLys Lys Ser Ala Thr Pro Ser Pro Ile Asn 1475 1480 1485 Met Leu Thr LysLeu Ile Ile Val Leu Arg Val Ala Trp Arg Asn Arg 1490 1495 1500 Gly LysAla Pro Leu Ser Thr Pro Leu Ala Ser Phe Arg Cys Met Thr 1505 1510 15151520 Arg Lys Ala Gln Asp Asp Leu Arg Phe Glu Glu Asn Ile Asp Ala Phe1525 1530 1535 Ser Met Gly Gly Gly Gln Gln Gly Arg Gln Ser Pro Thr AsnGlu Gly 1540 1545 1550 Arg Gly Gln Gln Glu Leu Gly Asn Ser Ala Asp TrpAsn Ile Glu Thr 1555 1560 1565 Val Ile Asp Trp Arg Lys Ile Val Ser MetTyr Tyr Gln Ala Asn Gly 1570 1575 1580 Lys Leu Thr Asp Gly Arg Thr LysGlu Asp Val Asp Leu Ala Met Ala 1585 1590 1595 1600 Val Pro Thr Ser PheIle Lys Pro Gln Gly Pro Asp Thr Thr Cys Arg 1605 1610 1615 Pro Ile AspTyr Thr Trp Leu Arg Leu Cys Lys Thr Lys Ser His Gly 1620 1625 1630 SerGly Leu Ser Ile Val Arg Arg Lys Thr Arg Gly Lys Ile Val Tyr 1635 16401645 Ser Thr Arg Thr Asn Thr Ser Val Leu Gln Ile Asn Ser Ser Arg Asn1650 1655 1660 Ala Pro Lys Ile Tyr Leu Arg Tyr Gly Arg Ala Lys Ile AlaHis Phe 1665 1670 1675 1680 Phe Phe Thr Ser Thr Thr Leu Lys Gly Gly AlaPhe Met Trp His Gly 1685 1690 1695 Leu Ala Ala Arg Leu Cys Lys Ile ArgVal Asp His Met 1700 1705 7 12 PRT Artificial Sequence Description ofArtificial Sequenceamino acid sequence conserved between Drosophila andC. elegans encoding degenerate primer sets 7 Leu Asp Val Leu Ile Glu AsnGlu Gln Lys Glu Val 1 5 10 8 11 PRT Artificial Sequence Description ofArtificial Sequenceamino acid sequence conserved between Drosophila andC. elegans encoding degenerate primer sets 8 His His Leu Phe Gly Pro TrpAla Ile Ile Ile 1 5 10 9 18 PRT Artificial Sequence Description ofArtificial Sequenceamino acid sequence conserved between Drosophila andC. elegans encoding degenerate primer sets 9 Val Leu Ile Asn Leu Leu IleAla Met Met Ser Asp Thr Tyr Gln Arg 1 5 10 15 Ile Gln 10 19 PRTArtificial Sequence Description of Artificial SequencenompCtransmembrane domain (channel region) #1 10 Ile Leu Leu Leu Leu Val AlaPhe Ile Val Cys Pro Pro Val Trp Ile 1 5 10 15 Gly Phe Thr 11 20 PRTArtificial Sequence Description of Artificial SequencenompCtransmembrane domain (channel region) #2 11 Tyr Trp Tyr Glu Val Gly LeuLeu Ile Trp Leu Ser Gly Leu Leu Leu 1 5 10 15 Phe Glu Leu Thr 20 12 20PRT Artificial Sequence Description of Artificial SequencenompCtransmembrane domain (channel region) #3 12 Ile Lys Val Leu Val Leu LeuLeu Gly Met Ala Gly Val Gly Val His 1 5 10 15 Val Ser Ala Phe 20 13 25PRT Artificial Sequence Description of Artificial SequencenompCtransmembrane domain (channel region) #4 13 Thr Leu Val Tyr Cys Arg AsnGln Cys Phe Ala Leu Ala Phe Leu Leu 1 5 10 15 Ala Cys Val Gln Ile LeuAsp Phe Leu 20 25 14 20 PRT Artificial Sequence Description ofArtificial SequencenompC transmembrane domain (channel region) #5 14 PheLeu Ala Val Leu Ala Ile Phe Val Phe Gly Phe Ser Met His Ile 1 5 10 15Val Ala Leu Asn 20 15 23 PRT Artificial Sequence Description ofArtificial SequencenompC transmembrane domain (channel region) #6 15 IleVal Phe Gly Ile Tyr Met Leu Val Ser Val Val Val Leu Ile Asn 1 5 10 15Leu Leu Ile Ala Met Met Ser 20 16 17 PRT Artificial Sequence Descriptionof Artificial SequencenompC transmembrane domain (channel region) #7 16Tyr Ile Asn Phe Ile Leu His Cys Val Leu Ile Ile Leu Tyr Phe Ser 1 5 1015 Ile 17 19 PRT Artificial Sequence Description of ArtificialSequencenompC transmembrane domain (channel region) #8 17 Ile Tyr LeuMet Ile His Leu Ser Ile Val Gly Ile Thr Pro Ile Tyr 1 5 10 15 Pro ValLeu

What is claimed is:
 1. An isolated nucleic acid encoding amechanosensory transduction protein, the protein having at least one ofthe following characteristics: (i) comprising greater than about 70%amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically bindingto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; wherein the proteindoes not comprise the amino acid sequence of SEQ ID NO:6.
 2. Theisolated nucleic acid of claim 1, wherein the nucleic acid encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4.
 3. The isolated nucleic acid of claim 1, wherein the nucleic acidcomprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 but notSEQ ID NO:5.
 4. The isolated nucleic acid of claim 1, wherein thenucleic acid selectively hybridizes under moderately stringent washconditions to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:3.
 5. The isolated nucleic acid of claim 1, whereinthe nucleic acid selectively hybridizes under stringent wash conditionsto a nucleic acid comprising a nucleotide sequence of SEQ ID NO:1 or SEQID NO:3 but not SEQ ID NO:5.
 6. The isolated nucleic acid of claim 1,wherein the nucleic acid is amplified by primers that selectivelyhybridize under stringent wash conditions to a nucleic acid having thesame sequence as degenerate primer sets encoding an amino acid sequenceselected from the group consisting of: LDVLIENEQKEV (SEQ ID NO:7);HHLFGPWAIII (SEQ ID NO:8); and VLINLLIAMMSDTYQRIQ (SEQ ID NO:9).
 7. Anexpression cassette comprising the nucleic acid of claim
 1. 8. Anisolated eukaryotic cell comprising the expression cassette of claim 7.9. An isolated nucleic acid encoding an extracellular domain of amechanosensory transduction protein, the extracellular domain comprisinggreater than about 70% amino acid sequence identity to an extracellulardomain of SEQ ID NO:2 or SEQ ID NO:4, wherein the extracellular domaindoes not comprise an extracellular domain of SEQ ID NO:6.
 10. Thenucleic acid of claim 7, wherein the extracellular domain is fused to aheterologous polypeptide, forming a chimeric polypeptide.
 11. Thenucleic acid of claim 8, wherein the extracellular domain comprises theamino acid sequence of an extracellular domain of SEQ ID NO:2 or SEQ IDNO:4.
 12. An isolated mechanosensory transduction protein, the proteinhaving at least one of the following characteristics: (i) comprisinggreater than about 70% amino acid sequence identity to SEQ ID NO:2 orSEQ ID NO:4; (ii) comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii)specifically binding to polyclonal antibodies generated against apolypeptide comprising an amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4; wherein the protein does not comprise the amino acid sequence ofSEQ ID NO:6.
 13. The isolated mechanosensory transduction protein ofclaim 12, wherein the protein comprises the amino acid sequence of SEQID NO:2 or SEQ ID NO:4.
 14. An isolated polypeptide comprising anextracellular domain of a mechanosensory transduction protein, theextracellular domain comprising greater than about 70% amino acidsequence identity to an extracellular domain of SEQ ID NO:2 or SEQ IDNO:4, wherein the extracellular domain does not comprise the amino acidsequence of an extracellular domain of SEQ ID NO:6.
 15. The isolatedpolypeptide of claim 14, wherein the extracellular domain is fused to aheterologous polypeptide, forming a chimeric polypeptide.
 16. Theisolated polypeptide of claim 14, wherein the extracellular domaincomprises the amino acid sequence of an extracellular domain of SEQ IDNO:2 or SEQ ID NO:4.
 17. An antibody that selectively binds to amechanosensory transduction protein, the protein having at least one ofthe following characteristics: (i) comprising greater than about 70%amino acid sequence identity to SEQ ID NO:2 or SEQ ID NO:4; (ii)comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (iii) specifically bindingto polyclonal antibodies generated against a polypeptide comprising anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4; wherein the proteindoes not comprise the amino acid sequence of SEQ ID NO:6.
 18. A methodfor identifying a compound that modulates mechanosensory receptoractivity in eukaryotic cells, the method comprising the steps of: (i)contacting the compound with a mechanosensory receptor protein, theprotein having at least one of the following characteristics: (a)comprising greater than about 70% amino acid sequence identity to asequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,and SEQ ID NO:6; (b) comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9; or (c)specifically binding to polyclonal antibodies generated against apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; and (ii)determining the functional effect of the compound on the mechanosensoryreceptor protein.
 19. The method of claim 18, wherein the mechanosensoryreceptor protein is expressed in a eukaryotic cell or cell membrane. 20.The method of claim 19, wherein the functional effect is determined bydetecting a change in the mechanoreceptor potential of the cell or cellmembrane.
 21. The method of claim 19, wherein the functional effect isdetermined by detecting a change in an intracellular ion concentration.22. The method of claim 21, wherein the ion is selected from the groupconsisting of K⁺ and Ca²⁺.
 23. The method of claim 18, wherein theprotein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
 24. The methodof claim 18, wherein the protein is recombinant.
 25. The method of claim18, wherein the functional effect is a physical interaction with thereceptor protein.
 26. A method of genotyping a human for amechanosensory transduction channel locus, the method comprisingdetecting a mutation in a nucleic acid encoding a mechanosensorytransduction protein in the human, the protein having at least one ofthe following characteristics: (a) comprising greater than about 70%amino acid sequence identity to a sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; (b) comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:7, SEQ ID NO:8, and SEQ ID NO:9; or (c) specifically binding topolyclonal antibodies generated against a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, and SEQ ID NO:6; wherein the mutation introduces apremature stop codon into the nucleic acid upstream of the regionencoding the transmembrane domain region of the protein, or is amissense mutation removing a cysteine residue between transmembranesegments 4 and 5 of the protein.